Novel anti-a2ap antibodies and uses thereof

ABSTRACT

The present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP. The isolated antibody or antigen-binding fragment according to the present invention i) cross-reacts with rabbit and/or cynomolgus A2AP, ii) does not inhibit human plasmin activity, and iii) increases plasmin mediated clot lysis in the presence of A2AP.

FIELD OF THE INVENTION

The present invention provides isolated antibodies or antigen-bindingfragments thereof that bind to human alpha 2 antiplasmin (A2AP). Theisolated antibodies or antigen-binding fragments according to thepresent invention i) cross-react with rabbit and/or cynomolgus A2AP, ii)do not bind to human plasmin/do not inhibit human plasmin activity, iii)do not convert A2AP from a serine protease inhibitor to a serineprotease substrate, iv) bind to human A2AP of the sequence of amino acid40-491 of SEQ ID 1 with a dissociation constant (KD)≤100 nM, ≤50 nM, ≤25nM, ≤10 nM, ≤1 nM, or ≤0.5 nM; v) bind to human A2AP of the sequence ofamino acid 40-491 of SEQ ID 1 with an EC50 of ≤500 nM, ≤250 nM, ≤100 nM,≤50 nM, ≤25 nM, ≤10 nM, ≤1 nM, or ≤0.5 nM; vi) inhibit the activity ofhuman A2AP of amino acid 40-491 of SEQ ID 1 with an EC50 of ≤500 nM,≤250 nM, ≤100 nM, ≤50 nM, ≤25 nM, ≤10 nM, ≤1 nM, or ≤0.5 nM; and/orincrease plasmin mediated clot lysis in the presence of A2AP.

The present invention further provides isolated nucleic acid sequencesencoding said antibodies or antigen-binding fragments and vectorscomprising same, isolated cells expressing said antibodies orantigen-binding fragments, methods of producing said antibodies orantigen-binding fragments and pharmaceutical compositions and kitscomprising said antibodies or antigen-binding fragments.

Antibodies according to the present invention can be used in thetreatment of diseases associated with ischemic events due to partial orcomplete vessel occlusion such as ischemic stroke, acute coronarysyndrome, peripheral artery disease, myocardial infarction, deep veinthrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis.

BACKGROUND OF THE INVENTION

Clot formation inside blood vessels can cause multiple severe diseaseslike ischemic stroke, acute coronary syndrome, peripheral arterydisease, myocardial infarction, deep vein thrombosis, pulmonaryembolism, venous thrombosis, or shunt thrombosis. Clot generation andpersistency are influenced by the rates of its formation by fibrin andplatelets and its dissolution by the lytic system. The current standardof care focusses on anticoagulation for chronic prevention ofthrombosis, which is despite all improvements by non-vitamin Kantagonist oral anticoagulants (NOACs) still accompanied by a bleedingrisk and has only an indirect effect on clot resolution.

The primary therapeutic goal for patients suffering from ischemic orembolic events is the timely restoration of blood flow. Reperfusiontherapy using thrombolysis, including intravenous (IV) recombinanttissue plasminogen activator (tPA) and endovascular interventions suchas mechanical thrombectomy (MT), are the only approved treatments forpatients suffering e.g. from ischemic stroke. However, both treatmentoptions have limitations. Especially for the short-acting tPA, its useis limited due to a strong increase in bleeding risk, neurotoxic effectsand limited time window of efficacy.

In contrast, inhibition of the major endogenous plasmin inhibitoralpha2-Antiplasmin (a2Ap) has been reported to improve pathologicalparameters without accompanying hemorrhage risk. Therefore, theinhibition of alpha2-Antiplasmin might be an innovative therapeuticoption for the acceleration of clot lysis and for the prevention of(secondary) thrombotic events.

Alpha2-Antiplasmin is a member of the Serpin superfamily. It is theprimary physiological inhibitor of the serine protease plasmin. Plasminin turn is an important enzyme that participates in fibrinolysis anddegradation of various other proteins. (Tone M, Kikuno R, Kume-Iwaki A,Hashimoto-Gotoh T. Structure of human alpha 2-plasmin inhibitor deducedfrom the cDNA sequence. J Biochem. 1987; 102(5):1033-1041; Silverman GA, Bird P I, Carrell R W, et al. The serpins are an expandingsuperfamily of structurally similar but functionally diverse proteins.Evolution, mechanism of inhibition, novel functions, and a revisednomenclature. J Biol Chem. 2001; 276(36):33293-33296).

Alpha2-Antiplasmin is synthesized as a 491 amino acid precursor with a27 amino acid signal peptide. The secreted form exhibits a shortpro-peptide (residues 28-39) and a mature chain (residues 40-491). Liverand kidney are major sites of a2Ap production, but also other tissuessuch as muscle, intestine, central nervous system, and placenta alsoexpress its mRNA at a moderate level.

Plasma concentrations of alpha2-Antiplasmin are ca. 1 micromolar (˜70micrograms/ml), the half-life in plasma is determined with 2.6 days.(Collen D, Wiman B. Turnover of antiplasmin, the fast-acting plasmininhibitor of plasma. Blood. 1979; 53(2):313-324).

Experimental therapeutic inactivation of a2-antiplasmin markedly reducesmicrovascular thrombosis, ischemic brain injury, brain swelling, brainhemorrhage and death after thromboembolic stroke (Reed G L, Houng A K,Wang D. Microvascular thrombosis, fibrinolysis, ischemic injury, anddeath after cerebral thromboembolism are affected by levels ofcirculating a2-antiplasmin. Arterioscler Thromb Vasc Biol. 2014;34(12):2586-2593).

Mimuro et al. describes JPTI-1, an A2AP antibody. The avidity of JPTI-1to preformed A2AP-plasmin-complex was lower than to free A2AP. JPTI-1inhibited A2AP activity by interfering with the formation ofA2AP-plasmin-complex. However, Mimuro et al is silent about the use ofJPTI-1 to enhance clot lysis (Mimuro, J. et al. Blood 1987; 69:446-453).

One of the best characterized known antibodies of the prior art is 77A3described by Reed et al. (Reed G L. Functional characterization ofmonoclonal antibody inhibitors of alpha 2-antiplasmin that acceleratefibrinolysis in different animal plasmas. Hybridoma. 1997;16(3):281-286; WO 98/12334, WO 98/12329), an antibody derived from aclassical mouse immunization approach. Due to the fact that thisantibody is of murine origin, at least a humanization campaign has beenperformed.

Other function blocking anti-alpha2-antiplasmin antibodies are notsuitable as therapeutic agents, either due to their origin as non-humanantibodies, e.g. the Serpin F2/alpha 2-Antiplasmin Antibody derived fromgoat (R&D, catalog number AF1484-SP), due to their specificity, e.g. themouse specific antibodies clone 27C9, 4H9, and CBYY-I0956, respectively(MyBioSource, catalogue numbers MBS135095 and MBS135076, CreativeBiolabs, catalogue number CBMAB-I2124-YY), or due the fact that theseantibodies are polyclonal (Invitrogen, catalogue number PAS-47142).

Thus, there exists a great need for novel therapeutic A2AP antibodiesuseful for the treatment of diseases that are associated with ischemicevents due to partial or complete vessel occlusion that has not been metso far.

OBJECTS OF THE INVENTION

In view of the prior art, it is an object of the present invention toprovide novel therapeutic A2AP antibodies that overcome the shortcomingof A2AP antibodies of the prior art. In particular it is an object ofthe present invention to provide novel A2AP antibodies that are highaffinity binders of human A2AP. Desirable A2AP antibodies arecross-reactive to rabbit and/or cynomolgus A2AP. They arenon-immunogenic in human therapy, i.e. they are human or humanizedantibodies. Desirable A2AP antibodies are selective to A2AP, inparticular they do not bind to and inhibit human plasmin. And they areable to increase plasmin mediated clot lysis in the presence of A2AP.

Such novel A2AP antibodies would offer major advances in the treatmentof diseases associated with ischemic events due to partial or completevessel occlusion such as ischemic stroke, acute coronary syndrome,peripheral artery disease, myocardial infarction, deep vein thrombosis,pulmonary embolism, venous thrombosis or shunt thrombosis.

SUMMARY OF THE INVENTION

The above-mentioned object and other objects are achieved by theteaching of the present invention. The present invention is based on thediscovery of novel antibodies that have a specific affinity foralpha2-Antiplasmin and can deliver a therapeutic benefit to a subject.

Thus, in a first aspect, the present invention relates to isolatedantibodies or antigen-binding fragments thereof that bind to human A2AP,wherein said isolated antibodies or antigen-binding fragments thereof

-   -   i) cross-react with rabbit and/or cynomolgus A2AP,    -   ii) do not inhibit human plasmin activity,    -   iii) do not convert A2AP from a serine protease inhibitor to a        serine protease substrate,    -   iv) bind to human A2AP of the sequence of amino acid 40-491 of        SEQ ID 1 with a dissociation constant (KD)≤100 nM, ≤50 nM, ≤25        nM, ≤10 nM, ≤1 nM, or ≤0.5 nM;    -   v) bind to human A2AP of the sequence of amino acid 40-491 of        SEQ ID 1 with an EC50 of ≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM, ≤25        nM, ≤10 nM, ≤1 nM, or ≤0.5 nM;    -   vi) inhibit the activity of human A2AP of amino acid 40-491 of        SEQ ID 1 with an EC50 of ≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM, ≤25        nM, ≤10 nM, ≤1 nM, or ≤0.5 nM; and/or increase plasmin mediated        clot lysis in the presence of A2AP.

The isolated antibodies or antigen-binding fragments according to thepresent invention are function blocking anti-alpha2-Antiplasminantibodies or antigen-binding fragments, which induce accelerated clotlysis in vitro as well as in vivo without leading to unwanted sideeffects like bleeding as it is typical for other pro-thrombolyticcompounds. Thus, the isolated antibodies or antigen-binding fragmentsaccording to the present invention may be used in the treatment ofdiseases associated with ischemic events due to vessel partial orcomplete occlusion such as ischemic stroke, acute coronary syndrome,peripheral artery disease, myocardial infarction, deep vein thrombosis,pulmonary embolism, venous thrombosis or shunt thrombosis. The isolatedantibodies or antigen-binding fragments according to the presentinvention may further be used in the diagnosis of A2AP-relateddisorders.

In a further aspect, the present invention relates to isolatedantibodies or antigen-binding fragments thereof capable of binding toA2AP and inhibiting the activity of A2AP, wherein said isolatedantibodies or antigen-binding fragments thereof do not inhibit plasminactivity.

In a further aspect, the present invention relates to isolatedantibodies or antigen-binding fragments thereof capable of binding tohuman A2AP and inhibiting activity of A2AP,

-   -   wherein said isolated antibodies or antigen-binding fragments        thereof bind to an epitope of A2AP comprising amino acid 402-408        (SRMSLSS) of SEQ ID NO: 1.

In a further aspect, the present invention relates to isolatedantibodies or antigen-binding fragments thereof which compete with saidisolated antibodies or antigen-binding fragments for binding to A2AP.

In a further aspect, the present invention relates to antibodyconjugates, comprising the isolated antibodies or antigen bindingfragments according to the invention.

In a further aspect, the present invention relates to isolated nucleicacid sequences that encode the antibodies or antigen-binding fragmentsaccording to the present invention.

In a further aspect, the present invention relates to vectors comprisinga nucleic acid sequence according to the present invention.

In a further aspect, the present invention relates to isolated cellsexpressing the antibodies or antigen-binding fragments according to thepresent invention and/or comprising the nucleic acid according to thepresent invention or the vector according to the present invention.

In a further aspect, the present invention relates to methods ofproducing the isolated antibodies or antigen-binding fragments accordingto the present invention comprising culturing of the cells according tothe present invention and optionally purification of the antibody orantigen-binding fragment.

In a further aspect, the present invention relates to pharmaceuticalcompositions comprising the isolated antibodies or antigen-bindingfragments according to the present invention or the antibody conjugatesaccording to the present invention.

In a further aspect, the present invention relates to isolatedantibodies or antigen-binding fragments according to the invention orconjugates according to invention or pharmaceutical compositionsaccording to the invention for use in the treatment or prophylaxis of adisease.

In a further aspect, the present invention relates to isolatedantibodies or antigen-binding fragments according to the invention orconjugates according to invention for use as a diagnostic agent.

In a further aspect, the present invention relates to isolatedantibodies or antigen-binding fragments according to the invention orconjugates according to invention or the pharmaceutical compositionaccording to the invention for use in the treatment or prophylaxis ofdisorders or diseases associated with ischemic events due to partial orcomplete vessel occlusion, such as ischemic stroke, acute coronarysyndrome, peripheral artery disease, myocardial infarction, deep veinthrombosis, pulmonary embolism, venous thrombosis, or shunt thrombosis.

In a further aspect, the present invention relates isolated antibodiesor antigen-binding fragments according to the invention or conjugatesaccording to invention or the pharmaceutical composition according tothe invention for use in simultaneous, separate, or sequentialcombination with one or more further therapeutically active compounds,particularly selected from inhibitors of the coagulation cascade,anticoagulants and platelet aggregation inhibitors.

In a further aspect, the present invention relates to kits comprisingthe isolated antibodies or antigen-binding fragments according to thepresent invention or the conjugates according to the present inventionand instructions for use.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the invention and the examplesincluded therein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs. The following references, however, canprovide one of skill in the art to which this invention pertains with ageneral definition of many of the terms used in this invention, and canbe referenced and used so long as such definitions are consistent withthe meaning commonly understood in the art. Such references include, butare not limited to, Singleton et al., Dictionary of Microbiology andMolecular Biology (2nd ed. 1994); The Cambridge Dictionary of Scienceand Technology (Walker ed., 1988); Hale & Marham, The Harper CollinsDictionary of Biology (1991); and Lackie et al., The Dictionary of Cell& Molecular Biology (3d ed. 1999); and Cellular and MolecularImmunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, W.B. SaundersCompany. Any additional technical resource available to the person ofordinary skill in the art providing definitions of terms used hereinhaving the meaning commonly understood in the art can be consulted. Forthe purposes of the present invention, the following terms are furtherdefined. Additional terms are defined elsewhere in the description. Asused herein and in the appended claims, the singular forms “a,” and“the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a gene” is a reference toone or more genes and includes equivalents thereof known to thoseskilled in the art, and so forth.

In the context of the present invention, the term “comprises” or“comprising” means “including, but not limited to”. The term is intendedto be open-ended, to specify the presence of any stated features,elements, integers, steps or components, but not to preclude thepresence or addition of one or more other features, elements, integers,steps, components or groups thereof. The term “comprising” thus includesthe more restrictive terms “consisting of” and “essentially consistingof”. In one embodiment the term “comprising” as used throughout theapplication and in particular within the claims may be replaced by theterm “consisting of”.

In this context, the term “about” or “approximately” means within 80% to120%, alternatively within 90% to 110%, including within 95% to 105% ofa given value or range.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

As used herein “A2AP” designates “alpha2-antiplasmin”, also known as“SerpinF2” (serpin family F member 2), AAP, API, PLI, or ALPHA-2-PI.A2AP is a member of the Serpin superfamily. It is the primaryphysiological inhibitor of the serine protease plasmin. A2AP issynthesized as a 491 amino acid precursor with a 27 amino acid signalpeptide. The secreted form exhibits a short pro-peptide (residues 28-39)and a mature chain (residues 40-491). A reference sequence for humanA2AP is available from UniProtKB/Swiss-Prot data base under accessionnumber P08697-1 (SEQ-ID NO:1), including signal peptide (positions1-27), pro-peptide (residues 28-39) and a mature chain (residues 40-491)(numbering is according to methionine in position 1).

Human A2AP (SEQ ID NO: 1):MALLWGLLVLSWSCLQGPCSVFSPVSAMEPLGRQLTSGPNQEQVSPLTLLKLGNQEPGGQTALKSPPGVCSRDPTPEQTHRLARAMMAFTADLESLVAQTSTCPNLILSPLSVALALSHLALGAQNHTLQRLQQVLHAGSGPCLPHLLSRLCQDLGPGAFRLAARMYLQKGFPIKEDFLEQSEQLFGAKPVSLTGKQEDDLANINQWVKEATEGKIQEFLSGLPEDTVLLLLNAIHFQGFWRNKFDPSLTQRDSFHLDEQFTVPVEMMQARTYPLRWELLEQPEIQVAHFPFKNNMSFVVLVPTHFEWNVSQVLANLSWDTLHPPLVWERPTKVRLPKLYLKHQMDLVATLSQLGLQELFQAPDLRGISEQSLVVSGVQHQSTLELSEVGVEAAAATSIAMSRMSLSSFSVNRPFLFFIFEDTTGLPLFVGSVRNPNPSAPRELKEQQDSPGNKDFLQSLKGFPRGDKLFGPDLKLVPPMEEDYPQFGSPK

Positions on Human A2AP domains SEQ-ID NO: 1 reactive center loop (RCL)400-412 arginine residue forming a covalent bond R403 with the activesite serine of plasmin C-terminal sequence including lysines 414-491interact with the kringle domains of plasmin

As used herein “plasmin” designates “plasmin”. Plasmin is a that acts todissolve fibrin e.g. in blood clots. Plasmin is released as a proenzymecalled plasminogen (PLG) from the liver into the systemic circulation.Two major glycoforms of plasminogen are present in humans—type Iplasminogen contains two glycosylation moieties (N-linked to N289 andO-linked to T346), whereas type II plasminogen contains only a singleO-linked sugar (O-linked to T346). Type II plasminogen is preferentiallyrecruited to the cell 25 surface over the type I glycoform. Conversely,type I plasminogen appears more readily recruited to blood clots. Incirculation, plasminogen adopts a closed, activation-resistantconformation. Upon binding to clots, or to the cell surface, plasminogenadopts an open form that can be converted into active plasmin by avariety of enzymes, including e.g. tissue plasminogen activator (tPA).Fibrin is a cofactor for plasminogen activation by tissue plasminogenactivator. The conversion of plasminogen to plasmin involves thecleavage of the peptide bond between Arg-561 and Val-562 (Wikipedia).

A reference sequence for human plasmin is available fromUniProtKB/Swiss-Prot data base under accession number P00747-1(numbering is according to methionine in position 1).

Positions on P00747-1 Human (numbering is according to plasminogendomain methionine in position 1). Plasmin  98-810 plasmin heavy chain A98-580 (SEQ ID 118) plasmin light chain B 581-810 (SEQ ID NO: 119)Kringle domain 1-5 103-560 Reactive site S741

The terms “anti-A2AP antibody” or “anti-alpha2-Antiplasmin antibody” and“an antibody that binds to alpha2-Antiplasmin” or “an antibody thatbinds to A2AP” refer to an antibody that is capable of bindingalpha2-Antiplasmin with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targetingalpha2-Antiplasmin. In one embodiment, the extent of binding of an antialpha2-Antiplasmin antibody to an unrelated, non-alpha2-Antiplasminprotein is less than about 10%, less than about 5%, or less than about2% of the binding of the antibody to alpha2-Antiplasmin as measured,e.g., by standard ELISA procedure. In certain embodiments, an antibodythat binds to alpha2-Antiplasmin has a binding activity (EC50) of ≤1 μM,≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M orless, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). Incertain embodiments, an anti-alpha2-Antiplasmin antibody binds to anepitope of alpha2-Antiplasmin that is conserved among alpha2-Antiplasminfrom different species.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules. Antibodies may comprise four polypeptidechains, two heavy (H) chains (about 50-70 kDa) and two light (L) chains(about 25 kDa) which are typically inter-connected by disulfide bonds.In particular embodiments, the antibody is composed of two identicalpairs of polypeptide chains. The amino-terminal portion of each chainincludes a “variable” region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The heavy chain variableregion is abbreviated herein as VH, the light chain variable region isabbreviated herein as VL. The carboxyl-terminal portion of each chaindefines a constant region primarily responsible for effector function.The heavy chain constant region can comprise e.g. three domains CH1, CH2and CH3. The light chain constant region is comprised of one domain(CL). The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is typically composed of three CDRs and upto four FRs, arranged from amino-terminus to carboxy-terminus e.g., inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, the term “Complementarity Determining Regions” (CDRs;e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDRs identified asCDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (e.g. about residues 23-36 (L1), 52-58 (L2) and91-101 (L3) in the light chain variable domain and 31-35 (H1), 50-65(H2) and 98-110 (H3) in the heavy chain variable domain; (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, MD. (1991)) and/orthose residues from a “hypervariable loop” (e.g. about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR defined according to Kabat and a hypervariable loop.

“Framework” or FR residues are those variable domain residues other thanthe hypervariable region residues.

The phrase “constant region” refers to the portion of the antibodymolecule that confers effector functions.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD,1991.

Immunoglobulins can be assigned to different classes depending on theamino acid sequence of the constant domain of their heavy chains. Heavychains are classified as mu GO, delta (A), gamma (γ), alpha (a), andepsilon (c), and define the antibody's isotype as IgM, IgD, IgG, IgA,and IgE, respectively. In particular embodiments, the antibody accordingto the present invention is an IgG antibody. Several of these may befurther divided into subclasses or isotypes, e.g. IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2. In particular embodiments, the antibody accordingto the present invention is an IgG1, an IgG2, an IgG3 or an IgG4antibody, more particularly an IgG1 or an IgG4 antibody. Differentisotypes may have different effector functions. Human light chains areclassified as kappa (κ) and lambda (λ) light chains. Within light andheavy chains, the variable and constant regions are joined by a “J”region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)).

A “functional fragment” or “antigen-binding antibody fragment” of anantibody/immunoglobulin hereby is defined as a fragment of anantibody/immunoglobulin (e.g., a variable region of an IgG) that retainsthe antigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hyper variable region(s) of anantibody, e.g., the CDR1, -2, and/or -3 regions; however, the variable“framework” regions can also play an important role in antigen binding,such as by providing a scaffold for the CDRs. Preferably, the“antigen-binding region” comprises at least amino acid residues 4 to 103of the variable light (VL) chain and 5 to 109 of the variable heavy (VH)chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111of VH, and particularly preferred are the complete VL and VH chains(amino acid positions 1 to 109 of VL and 1 to 113 of VH; numberingaccording to WO 97/08320).

Nonlimiting examples of “functional fragments” or “antigen-bindingantibody fragments” include Fab, Fab′, F(ab′)2, Fv fragments, domainantibodies (dAb), complementarity determining region (CDR) fragments,single-chain antibodies (scFv), single chain antibody fragments,diabodies, triabodies, tetrabodies, minibodies, linear antibodies(Zapata et al., Protein Eng., 8 (10): 1057-1062 (1995)); chelatingrecombinant antibodies, tribodies or bibodies, intrabodies, nanobodies,small modular immunopharmaceuticals (SMIPs), an antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or muteins or derivatives thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as a CDRsequence, as long as the antibody retains the desired biologicalactivity; and multispecific antibodies such as bi- and tri-specificantibodies formed from antibody fragments (C. A. K Borrebaeck, editor(1995) Antibody Engineering (Breakthroughs in Molecular Biology), OxfordUniversity Press; R. Kontermann & S. Duebel, editors (2001) AntibodyEngineering (Springer Laboratory Manual), Springer Verlag). An antibodyother than a “bispecific” or “bifunctional” antibody is understood tohave each of its binding sites identical. The F(ab′)2 or Fab may beengineered to minimize or completely remove the intermolecular disulfideinteractions that occur between the C_(H1) and C_(L) domains. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two “Fv” fragments. An “Fv” fragment is the minimum antibodyfragment that contains a complete antigen recognition and binding site.This region consists of a dimer of one heavy- and one light-chainvariable domain in tight, non-covalent association. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the VH-VL dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen.

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise the VHand VL domains of antibody, wherein these domains are present in asingle polypeptide chain.

Preferably, the Fv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains that enables the Fv to form the desiredstructure for antigen binding. For a review of Fvs see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994).

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine residues from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)2 antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteine residues between them.

The term “mutein” or “variant” can be used interchangeably and refers toan antibody or antigen-binding fragment that contains at least one aminoacid substitution, deletion, or insertion in the variable region or theportion equivalent to the variable region, provided that the mutein orvariant retains the desired binding affinity or biological activity.Variants of the antibodies or antigen-binding antibody fragmentscontemplated in the invention are molecules in which the bindingactivity of the antibody or antigen-binding antibody fragment ismaintained.

A “chimeric antibody” or antigen-binding fragment thereof is definedherein as one, wherein the variable domains are derived from a non-humanorigin and some or all constant domains are derived from a human origin.

“Humanized antibodies” contain CDR regions derived from a non-humanspecies, such as mouse, that have, for example, been engrafted, alongwith any necessary framework back-mutations, into human sequence-derivedV regions. Thus, for the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired specificity,affinity, and capacity. See, for example, U.S. Pat. Nos. 5,585,089;5,693,761; 5,693,762; 5,859,205, each herein incorporated by reference.In some instances, framework residues of the human immunoglobulin arereplaced by corresponding non-human residues (see, for example, U.S.Pat. Nos. 5,585,089; 5,693,761; 5,693,762, each herein incorporated byreference). Furthermore, humanized antibodies may comprise residues thatare not found in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the framework regions are those of a humanimmunoglobulin sequence. The humanized antibody optionally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details see Joneset al., Nature 331:522-25 (1986); Riechmann et al., Nature 332:323-27(1988); and Presta, Curr. Opin. Struct. Biol. 2:593-96 (1992), eachherein incorporated by reference.

“Human antibodies” or “fully human antibodies” comprise human derivedCDRs, i.e. CDRs of human origin. Fully human antibodies may comprise alow number of germline deviations compared with the closest humangermline reference determined based on the IMGT database(http://www.imgt.org). For example, a fully human antibody according tothe current invention may comprise up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10germline deviations in the CDRs compared with the closest human germlinereference. Fully human antibodies can be developed from human derived Bcells by cloning techniques in combination with a cell enrichment orimmortalization step. The majority of fully human antibodies, however,are isolated either from immunized mice transgenic for the human IgGlocus or from sophisticated combinatorial libraries by phage display(Bruggemann M., Osborn M. J., Ma B., Hayre J., Avis S., Lundstrom B. andBuelow R., Human Antibody Production in Transgenic Animals, Arch ImmunolTher Exp (Warsz.) 63 (2015), 101-108; Carter P. J., Potent antibodytherapeutics by design, Nat Rev Immunol 6 (2006), 343-357; Frenzel A.,Schirrmann T. and Hust M., Phage display-derived human antibodies inclinical development and therapy, MAbs 8 (2016), 1177-1194; Nelson A.L., Dhimolea E. and Reichert J. M., Development trends for humanmonoclonal antibody therapeutics, Nat Rev Drug Discov 9 (2010),767-774.)).

Several techniques are available to generate fully human antibodies (cf.WO2008/112640 A3). Cambridge Antibody Technologies (CAT) and Dyax haveobtained antibody cDNA sequences from peripheral B cells isolated fromimmunized humans and devised phage display libraries for theidentification of human variable region sequences of a particularspecificity. Briefly, the antibody variable region sequences are fusedeither with the Gene III or Gene VIII structure of the M13bacteriophage. These antibody variable region sequences are expressedeither as Fab or single chain Fv (scFv) structures at the tip of thephage carrying the respective sequences. Through rounds of a panningprocess using different levels of antigen binding conditions(stringencies), phages expressing Fab or scFv structures that arespecific for the antigen of interest can be selected and isolated. Theantibody variable region cDNA sequences of selected phages can then beelucidated using standard sequencing procedures. These sequences maythen be used for the reconstruction of a full antibody having thedesired isotype using established antibody engineering techniques.Antibodies constructed in accordance with this method are consideredfully human antibodies (including the CDRs). In order to improve theimmunoreactivity (antigen binding affinity and specificity) of theselected antibody, an in vitro maturation process can be introduced,including a combinatorial association of different heavy and lightchains, deletion/addition/mutation at the CDR3 of the heavy and lightchains (to mimic V-J, and V-D-J recombination), and random mutations (tomimic somatic hypermutation). An example of a “fully human” antibodygenerated by this method is the anti-tumor necrosis factor α antibody,Humira (adalimumab).

“Human Engineered™” antibodies generated by altering the parent sequenceaccording to the methods set forth in Studnicka et al., U.S. Pat. No.5,766,886.

An antibody of the invention may be derived from a recombinant antibodygene library. The development of technologies for making repertoires ofrecombinant human antibody genes, and the display of the encodedantibody fragments on the surface of filamentous bacteriophage, hasprovided a recombinant means for directly making and selecting humanantibodies, which also can be applied to humanized, chimeric, murine ormutein antibodies. The antibodies produced by phage technology areproduced as antigen binding fragments—usually Fv or Fab fragments—inbacteria and thus lack effector functions. Effector functions can beintroduced by one of two strategies: The fragments can be engineeredeither into complete antibodies for expression in mammalian cells, orinto bispecific antibody fragments with a second binding site capable oftriggering an effector function. Typically, the Fd fragment (VH-CH1) andlight chain (VL-CL) of antibodies are separately cloned by PCR andrecombined randomly in combinatorial phage display libraries, which canthen be selected for binding to a particular antigen. The Fab fragmentsare expressed on the phage surface, i.e., physically linked to the genesthat encode them. Thus, selection of Fab by antigen binding co-selectsfor the Fab encoding sequences, which can be amplified subsequently. Byseveral rounds of antigen binding and re-amplification, a proceduretermed panning, Fab specific for the antigen are enriched and finallyisolated.

A variety of procedures have been described for human antibodiesderiving from phage-display libraries. Such libraries may be built on asingle master framework, into which diverse in vivo-formed (i. e.human-derived) CDRs are allowed to recombine as described by Carlssonand Söderlind Exp. Rev. Mol. Diagn. 1 (1), 102-108 (2001), Söderlin etal., Nat. Biotech. 18, 852-856 (2000) and U.S. Pat. No. 6,989,250.Alternatively, such an antibody library may be based on amino acidsequences that have been designed in silico and encoded by nucleic acidsthat are synthetically created. In silico design of an antibody sequenceis achieved, for example, by analyzing a database of human sequences anddevising a polypeptide sequence utilizing the data obtained therefrom.Methods for designing and obtaining in silico-created sequences aredescribed, for example, in Knappik et al., J. Mol. Biol. (2000) 296:57;Krebs et al., J. Immunol. Methods. (2001) 254:67; and U.S. Pat. No.6,300,064. For a review of phage display screening (for example see HoetR M et al, Nat Biotechnol 2005; 23(3):344-8), the well-establishedhybridoma technology (for example see Kohler and Milstein Nature. 1975Aug. 7; 256(5517):495-7), or immunization of mice inter aliaimmunization of hMAb mice (e.g. VelocImmune Mouse®).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the term “monoclonal” indicatesthe character of the antibody as not being a mixture of discreteantibodies. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. In addition to their specificity, monoclonal antibodypreparations are advantageous in that they are typically uncontaminatedby other immunoglobulins. The term “monoclonal” is not to be construedas to require production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used may be made by thehybridoma method first described by Kohler et al., Nature, 256: 495[1975, or may be made by recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567). The “monoclonal antibodies” may also be recombinant,chimeric, humanized, human, Human Engineered™, or antibody fragments,for example.

An “isolated” antibody is one that has been identified and separatedfrom a component of the cell that expressed it. Contaminant componentsof the cell are materials that would interfere with diagnostic ortherapeutic uses of the antibody, and may include enzymes, hormones, andother proteinaceous or non-proteinaceous solutes.

An “isolated” nucleic acid is one that has been identified and separatedfrom a component of its natural environment. An isolated nucleic acidincludes a nucleic acid molecule contained in cells that ordinarilycontain the nucleic acid molecule, but the nucleic acid molecule ispresent extrachromosomally or at a chromosomal location that isdifferent from its natural chromosomal location.

As used herein, an antibody “binds specifically to”, is “specificto/for” or “specifically recognizes” an antigen of interest, e.g. A2AP,is one that binds the antigen with sufficient affinity such that theantibody is useful as a therapeutic agent in targeting a cell or tissueexpressing the antigen, and does not significantly cross-react withproteins other than orthologs and variants (e.g. mutant forms, splicevariants, or proteolytically truncated forms) of the aforementionedantigen target. The term “specifically recognizes” or “bindsspecifically to” or is “specific to/for” a particular polypeptide or anepitope on a particular polypeptide target as used herein can beexhibited, for example, by an antibody, or antigen-binding fragmentthereof, having a monovalent K_(D) for the antigen of less than about10⁻⁴ M, alternatively less than about 10⁻⁵ M, alternatively less thanabout 10⁻⁶ M, alternatively less than about 10⁻⁷ M, alternatively lessthan about 10⁻⁸ M, alternatively less than about 10⁻⁹ M, alternativelyless than about 10⁻¹⁰ M, alternatively less than about 10⁻¹¹ M,alternatively less than about 10⁻¹² M, or less. An antibody “bindsspecifically to,” is “specific to/for” or “specifically recognizes” anantigen if such antibody is able to discriminate between such antigenand one or more reference antigen(s). In its most general form,“specific binding”, “binds specifically to”, is “specific to/for” or“specifically recognizes” is referring to the ability of the antibody todiscriminate between the antigen of interest and an unrelated antigen,as determined, for example, in accordance with one of the followingmethods. Such methods comprise, but are not limited to surface plasmonresonance (SPR), Western blots, ELISA-, RIA-, ECL-, IRMA-tests andpeptide scans. For example, a standard ELISA assay can be carried out.The scoring may be carried out by standard color development (e.g.secondary antibody with horseradish peroxidase and tetramethyl benzidinewith hydrogen peroxide). The reaction in certain wells is scored by theoptical density, for example, at 450 nm. Typical background (=negativereaction) may be 0.1 OD; typical positive reaction may be 1 OD. Thismeans the difference positive/negative is more than 5-fold, 10-fold,50-fold, and preferably more than 100-fold. Typically, determination ofbinding specificity is performed by using not a single referenceantigen, but a set of about three to five unrelated antigens, such asmilk powder, BSA, transferrin or the like.

“Binding affinity” or “affinity” refers to the strength of the total sumof non-covalent interactions between a single binding site of a moleculeand its binding partner. Unless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a1:1 interaction between members of a binding pair (e.g. an antibody andan antigen). The dissociation constant “K D” is commonly used todescribe the affinity between a molecule (such as an antibody) and itsbinding partner (such as an antigen) i.e. how tightly a ligand binds toa particular protein. Ligand-protein affinities are influenced bynon-covalent intermolecular interactions between the two molecules.Affinity can be measured by common methods known in the art, includingthose described herein. In one embodiment, the “K_(D)” or “K_(D) value”according to this invention is measured by using surface plasmonresonance assays using a Biacore T200 instrument (GE Healthcare Biacore,Inc.). Other suitable devices are BIACORE T100, BIACORE®-2000, BIACORe4000, a BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ), or ProteOn XPR36instrument (Bio-Rad Laboratories, Inc.).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains, orcombinations thereof and usually have specific three-dimensionalstructural characteristics, as well as specific charge characteristics.

An “antibody that binds to the same epitope” as a reference antibody or“an antibody which competes for binding” to a reference antibody refersto an antibody that blocks binding of the reference antibody to itsantigen in a competition assay by 10%, 20%, 30%, 40%, 50% or more, andconversely, the reference antibody blocks binding of the antibody to itsantigen in a competition assay by 10%, 20%, 30%, 40%, 50% or more.

The term “maturated antibodies” or “maturated antigen-binding fragments”such as maturated Fab variants or “optimized” variants includesderivatives of an antibody or antibody fragment exhibiting strongerbinding—i. e. binding with increased affinity—to a given antigen such asthe extracellular domain of a target protein. Maturation is the processof identifying a small number of mutations within the six CDRs of anantibody or antibody fragment leading to this affinity increase. Thematuration process is the combination of molecular biology methods forintroduction of mutations into the antibody and screening foridentifying the improved binders.

“Percent (%) sequence identity” with respect to a referencepolynucleotide or polypeptide sequence, respectively, is defined as thepercentage of nucleic acid or amino acid residues, respectively, in acandidate sequence that are identical with the nucleic acid or aminoacid residues, respectively, in the reference polynucleotide orpolypeptide sequence, respectively, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Conservative substitutions are not considered as part of thesequence identity. Preferred are un-gapped alignments. Alignment forpurposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for aligning sequences, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

“Sequence homology” indicates the percentage of amino acids that eitheris identical or that represent conservative amino acid substitutions.

An “antagonistic” antibody or a “blocking” antibody is one whichsignificantly inhibits (either partially or completely) a biologicalactivity of the antigen it binds. In particular embodiments, antibodiesor antigen-binding fragments according to the present invention are anA2AP blocking antibodies or antigen-binding fragments.

The term “antibody conjugate” refers to an antibody conjugated to one ormore molecules including drugs—in which case the antibody conjugate isreferred to as “antibody-drug conjugate” (“ADC”)—and high molecularweight molecules such as peptides or proteins.

Amino acids may be referred to herein by their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which at least oneexogenous nucleic acid has been introduced, including the progeny ofsuch cells. Host cells include “transformants” and “transformed cells”,“transfectants” and “transfected cells” and “transduced cells” whichinclude the primary transformed/transfected/transduced cell and progenyderived therefrom without regard to the number of passages. Progeny maynot be completely identical in nucleic acid content to a parent cell butmay contain mutations. Mutant progeny that have the same function orbiological activity as screened or selected for in the originallytransformed cell are included herein.

As used herein, the phrase “therapeutically effective amount” is meantto refer to an amount of therapeutic or prophylactic antibody that wouldbe appropriate to elicit the desired therapeutic or prophylactic effector response, including alleviating some or all of such symptoms ofdisease or reducing the predisposition to the disease, when administeredin accordance with the desired treatment regimen.

The term “pharmaceutical formulation”/“pharmaceutical composition”refers to a preparation which is in such form as to permit thebiological activity of an active ingredient contained therein to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the formulation would beadministered.

Antibodies According to the Present Invention

In one aspect, the present invention relates to isolated antibodies orantigen-binding fragments thereof binding to human A2AP, wherein saidisolated antibodies or antigen-binding fragments thereof cross-reactwith rabbit and/or cynomolgus A2AP. In particular embodiments, theisolated antibodies or antigen-binding fragments according to thepresent invention have an affinity to rabbit A2AP that is less than100-fold, particularly less than 30-fold, even more particularly lessthan 15-fold and most particularly less than different to that to humanA2AP. In particular such embodiments, said affinities are to human A2APof amino acid 40-491 of SEQ ID NO: 1 and to rabbit A2AP of amino acids28-491 of SEQ ID NO: 2. In particular embodiments, the isolatedantibodies or antigen-binding fragments according to the presentinvention have an affinity to cynomolgus A2AP that is less than100-fold, particularly less than 30-fold, even more particularly lessthan 15-fold and most particularly less than 5-fold different to that tohuman A2AP. In particular such embodiments, said affinities are to humanA2AP of amino acid 40-491 of SEQ ID NO: 1 and to cynomolgus A2AP ofamino acid 28-491 SEQ ID NO: 3.

In another aspect, the present invention relates to isolated antibodiesor antigen-binding fragments thereof capable of binding to human A2APand inhibiting activity of A2AP, wherein said isolated antibodies orantigen-binding fragments thereof do not inhibit plasmin activity.

In particular embodiments, said isolated antibodies or antigen-bindingfragments thereof according to the present invention inhibit theactivity of A2AP by preventing the binding of A2AP to plasmin.

Due to the fact that the endogenous plasma concentration of A2AP iscomparatively high (1 μM; and 70 μg/ml, respectively), highconcentration of neutralizing antibody will be needed to block A2APactivity. Therefore, it is advantageous if an A2AP antibody does notinhibit plasmin activity up to high concentration of the antibody.Surprisingly, isolated antibodies or antigen-binding fragments thereofaccording to the present invention did not inhibit plasmin activity upto a concentration of 10 μM in an in vitro plasmin inhibition assay,whereas antibody 77A3 inhibited the plasmin activity with an IC50 of 1.7μM in the same assay (see example 11, FIG. 16 ).

In particular embodiments, said isolated antibodies or antigen-bindingfragments thereof according to the present invention do not inhibit theactivity of human plasmin, particularly human plasmin comprising SEQ IDNO: 118 (plasmin heavy chain A) and SEQ ID NO: 119 (plasmin light chainB), even if present in high micromolar concentration.

In particular embodiments, said isolated antibodies or antigen-bindingfragments thereof according to the present invention do not inhibitplasmin activity up to a concertation of said isolated antibodies orantigen-binding fragments thereof of 1 μM, 2 μM, 5 μM or 10 μM in an invitro plasmin inhibition assay.

In particular embodiments, said isolated antibodies or antigen-bindingfragments thereof according to the present invention do not inhibitplasmin activity up to a concertation of said isolated antibodies orantigen-binding fragments thereof of 1 μM, 2 μM, 5 μM or 10 μM in an invitro plasmin inhibition assay, wherein the in vitro plasmin inhibitionassay determines the inhibition of the proteolytic activity of plasmin.

Such an in vitro plasmin inhibition assay can be an assay, thatdetermines the inhibition of the proteolytic activity of plasmin asdescribed in example 11. For such assay plasmin and a labeled substratefor the plasmin proteolytic activity like fluorogenic substrate 1-1275(Bachem; MeOSuc-Ala-Phe-Lys-AMC trifluoroacetate salt; catalogue number1-1275) may be used.

In another aspect, the present invention relates to isolated antibodiesor antigen-binding fragments thereof capable of binding to human A2APand inhibiting activity of A2AP,

-   -   wherein said isolated antibodies or antigen-binding fragments        thereof bind to an epitope of A2AP comprising amino acid 402-408        (SRMSLSS) of SEQ ID NO: 1.

A2AP comprises the amino acid sequence SRMSLSS (amino acid 402-408 ofSEQ ID NO: 1), which is located in the reactive center loop of A2AP(amino acid 400-412 of Seq ID NO: 1). In particular embodiments, saidisolated antibodies or antigen-binding fragments thereof according tothe present invention are capable of binding to human A2AP andinhibiting activity of A2AP, wherein said isolated antibodies orantigen-binding fragments thereof bind to an epitope of A2AP comprisingamino acid 402-408 (SRMSLSS) of SEQ ID NO: 1 and wherein said isolatedantibodies or antigen-binding fragments thereof do not inhibit plasminactivity.

In particular embodiments, said isolated antibodies or antigen-bindingfragments thereof according to the present invention are capable ofbinding to human A2AP and inhibiting activity of A2AP, wherein saidisolated antibodies or antigen-binding fragments thereof bind to anepitope of A2AP comprising amino acid 402-408 (SRMSLSS) of SEQ ID NO: 1and wherein said isolated antibodies or antigen-binding fragmentsthereof do not inhibit plasmin activity up to a concertation of saidisolated antibodies or antigen-binding fragments thereof of 1 μM, 2 μM,5 μM or 10 μM in an in vitro plasmin inhibition assay.

In another aspect, the present invention relates to isolated antibodiesor antigen-binding fragments thereof binding to human A2AP, wherein saidisolated antibodies or antigen-binding fragments thereof do not convertA2AP from a serine protease inhibitor to a serine protease substrate.

In another aspect, the present invention relates to isolated antibodiesor antigen-binding fragments thereof binding to human A2AP, wherein saidisolated antibodies or antigen-binding fragments thereof bind to humanA2AP of the sequence of amino acid 40-491 of SEQ ID 1 with adissociation constant (KD)≤100 nM, ≤50 nM, ≤25 nM, ≤10 nM, ≤1 nM, or≤0.5 nM.

In another aspect, the present invention relates to isolated antibodiesor antigen-binding fragments thereof binding to human A2AP, wherein saidisolated antibodies or antigen-binding fragments thereof bind to humanA2AP of the sequence of amino acid 40-491 of SEQ ID 1 with an EC50 of≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM, ≤25 nM, ≤10 nM, ≤1 nM, or ≤0.5 nM.

In another aspect, the present invention relates to isolated antibodiesor antigen-binding fragments thereof binding to human A2AP, wherein saidisolated antibodies or antigen-binding fragments thereof inhibit theactivity of human A2AP of amino acid 40-491 of SEQ ID 1 with an EC50 of≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM, ≤25 nM, ≤10 nM, ≤1 nM, or ≤0.5 nM inan in vitro A2AP function blocking assay.

An in vitro A2AP function blocking assay can be an assay as described inexample 4. In such an assay, the test antibodies are pre-incubated withA2AP. After adding a A2AP-substrate like plasmin, trypsin orchymotrypsin to the assay, the activity of the added A2AP substrate,which is not blocked by A2AP, can be analyzed for example by the use ofa labeled substrate for the A2AP-substrate. For example, for plasmin(A2AP substrate) the fluorogenic substrate 1-1275 (Bachem;MeOSuc-Ala-Phe-Lys-AMC trifluoroacetate salt; catalogue number 1-1275)may be used.

In particular embodiments, said isolated antibodies or antigen-bindingfragments thereof according to the present invention

-   -   (i) bind to human A2AP of the sequence of amino acid 40-491 of        SEQ ID NO: 1 with a dissociation constant (KD)≤100 nM, ≤50 nM,        ≤25 nM, ≤10 nM, ≤1 nM, or ≤0.5 nM; and    -   (ii) inhibit the activity of human A2AP of amino acid 40-491 of        SEQ ID NO: 1 with an EC50 of ≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM,        ≤25 nM, ≤10 nM, ≤1 nM, or ≤0.5 nM in an in vitro A2AP function        blocking assay.

In another aspect, the present invention relates to isolated antibodiesor antigen-binding fragments thereof binding to human A2AP, wherein saidisolated antibodies or antigen-binding fragments thereof increaseplasmin mediated clot lysis in the presence of A2AP. In particular, theisolated antibodies or antigen-binding fragments according to thepresent invention increase plasmin mediated clot lysis in vitro and/orin vivo. The antibody's ability to increase plasmin mediated clot lysisin vitro may be assessed as described in Example 7. The antibody'sability to increase plasmin mediated clot lysis in vivo may be assessedas described in Example 8.

The isolated antibodies or antigen-binding fragments according to thepresent invention may exhibit any combination of the above describedcharacteristics.

In particular embodiments, the isolated antibodies or antigen-bindingfragments according to the present invention interfere with theinteraction of A2AP and plasmin, particularly with the interaction ofhuman A2AP and human plasmin, particularly with the interaction of humanA2AP of amino acid 40-491 of SEQ ID NO: 1 and human plasmin,particularly human plasmin comprising SEQ ID NO: 118 (plasmin heavychain A) and SEQ ID NO: 119 (plasmin light chain B). Particularly, theantibodies or antigen-binding fragments according to the presentinvention are A2AP blocking antibodies or antigen-binding fragments.

In particular embodiments, the isolated antibodies or antigen-bindingfragments according to the present invention comprise a heavy chainvariable domain that is at least 90%, at least 95%, at least 98% or atleast 99% identical to SEQ ID NO: 32. In particular embodiments, theisolated antibodies or antigen-binding fragments according to thepresent invention comprise a light chain variable domain that is atleast 90% %, at least 95%, at least 98% or at least 99% identical to SEQID NO: 38. In particular embodiments, the isolated antibodies orantigen-binding fragments according to the present invention comprise aheavy chain variable domain that is at least 90%, at least 95%, at least98% or at least 99% identical to SEQ ID NO: 32, and a light chainvariable domain that is at least 90% %, at least 95%, at least 98% or atleast 99% identical to SEQ ID NO: 38.

In particular embodiments, the isolated antibody or antigen-bindingfragment according to the present invention comprises a heavy chainantigen-binding region that comprises an H-CDR3 comprising the sequenceEX₁YDSSGYYHLX₂Y (SEQ ID NO: 4) wherein X₁ is selected from the groupconsisting of Y, D and G and wherein X₂ is selected from the groupconsisting of D, V, E and T. In particular embodiments, X₁ is selectedfrom the group consisting of D and G and X₂ is selected from the groupconsisting of V, E and T.

In particular embodiments, the isolated antibody or antigen-bindingfragment according to the present invention comprises a light chainantigen-binding region that comprises an L-CDR3 comprising the sequenceX₁AWDX₂SLSGWV (SEQ ID NO: 5) wherein X₁ is selected from the groupconsisting of A and W and wherein X₂ is selected from the groupconsisting of D, N, L, W and V. In particular embodiments, X₁ isselected from the group consisting of W and X₂ is selected from thegroup consisting of N, L, W and V.

Particularly, the isolated antibody or antigen-binding fragmentaccording to the present invention comprises i) a heavy chainantigen-binding region that comprises an H-CDR3 comprising the sequenceEX₁YDSSGYYHLX₂Y (SEQ ID NO: 4) wherein X₁ is selected from the groupconsisting of Y, D and G and wherein X₂ is selected from the groupconsisting of D, V, E and T and ii) a light chain antigen-binding regionthat comprises an L-CDR3 comprising the sequence X₁AWDX₂SLSGWV (SEQ IDNO: 5), wherein X₁ is selected from the group consisting of A and W; andwherein X₂ is selected from the group consisting of D, N, L, W and V.

In particular embodiments, the two framework residues X₁X₂ directlyadjacent to the 5′ end of the H-CDR3 region (corresponding to residues96 [X₁] and 97 [X₂] of reference VH domain of SEQ ID NO: 32) areselected as follows: X₁ is selected from the group consisting of A andD, in particular X₁ is D, and X₂ is selected from the group consistingof R and S, in particular X₂ is S.

In particular embodiments, the isolated antibody or antigen-bindingfragment according to the present invention comprises a heavy chainantigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6or SEQ ID NO: 21 and an H-CDR2 comprising SEQ ID NO: 7, SEQ ID NO: 8 orSEQ ID NO: 22. In particular embodiments, the isolated antibody orantigen-binding fragment according to the present invention comprises alight chain antigen-binding region that comprises an L-CDR1 comprisingSEQ ID NO: 9 and an L-CDR2 comprising SEQ ID NO: 10.

In particular embodiments, the isolated antibody or antigen-bindingfragment according to the present invention comprises

-   -   i) a heavy chain antigen-binding region that comprises an H-CDR3        comprising SEQ ID NO: 13 and a light chain antigen-binding        region that comprises an L-CDR3 comprising SEQ ID NO: 18, or    -   ii) a heavy chain antigen-binding region that comprises an        H-CDR3 comprising SEQ ID NO: 11 and a light chain        antigen-binding region that comprises an L-CDR3 comprising SEQ        ID NO: 17, or    -   iii) a heavy chain antigen-binding region that comprises an        H-CDR3 comprising SEQ ID NO: 11 and a light chain        antigen-binding region that comprises an L-CDR3 comprising SEQ        ID NO: 18, or    -   iv) a heavy chain antigen-binding region that comprises an        H-CDR3 comprising SEQ ID NO: 12 and a light chain        antigen-binding region that comprises an L-CDR3 comprising SEQ        ID NO: 18, or    -   v) a heavy chain antigen-binding region that comprises an H-CDR3        comprising SEQ ID NO: 13 and a light chain antigen-binding        region that comprises an L-CDR3 comprising SEQ ID NO: 19, or    -   vi) a heavy chain antigen-binding region that comprises an        H-CDR3 comprising SEQ ID NO: 13 and a light chain        antigen-binding region that comprises an L-CDR3 comprising SEQ        ID NO: 18, or    -   vii) a heavy chain antigen-binding region that comprises an        H-CDR3 comprising SEQ ID NO: 14 and a light chain        antigen-binding region that comprises an L-CDR3 comprising SEQ        ID NO: 18, or    -   viii) a heavy chain antigen-binding region that comprises an        H-CDR3 comprising SEQ ID NO: 14 and a light chain        antigen-binding region that comprises an L-CDR3 comprising SEQ        ID NO: 20, or    -   ix) a heavy chain antigen-binding region that comprises an        H-CDR3 comprising SEQ ID NO: 15 and a light chain        antigen-binding region that comprises an L-CDR3 comprising SEQ        ID NO: 18, or    -   x) a heavy chain antigen-binding region that comprises an H-CDR3        comprising SEQ ID NO: 16 and a light chain antigen-binding        region that comprises an L-CDR3 comprising SEQ ID NO: 18.

In particular embodiments, the isolated antibodies or antigen-bindingfragments according to the present invention comprises:

-   -   i) a heavy chain antigen-binding region that comprises an H-CDR1        comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 8, and        an H-CDR3 comprising SEQ ID NO: 13 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:18, or    -   ii) a heavy chain antigen-binding region that comprises an        H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO:        7, and an H-CDR3 comprising SEQ ID NO: 13 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:18, or    -   iii) a heavy chain antigen-binding region that comprises an        H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO:        7, and an H-CDR3 comprising SEQ ID NO: 11 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:17, or    -   iv) a heavy chain antigen-binding region that comprises an        H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO:        7, and an H-CDR3 comprising SEQ ID NO: 11 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:18, or    -   v) a heavy chain antigen-binding region that comprises an H-CDR1        comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and        an H-CDR3 comprising SEQ ID NO: 12 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:18, or    -   vi) a heavy chain antigen-binding region that comprises an        H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO:        7, and an H-CDR3 comprising SEQ ID NO: 13 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:19, or    -   vii) a heavy chain antigen-binding region that comprises an        H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO:        7, and an H-CDR3 comprising SEQ ID NO: 14 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:18, or    -   viii) a heavy chain antigen-binding region that comprises an        H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO:        7, and an H-CDR3 comprising SEQ ID NO: 14 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:20, or    -   ix) a heavy chain antigen-binding region that comprises an        H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO:        7, and an H-CDR3 comprising SEQ ID NO: 15 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:18, or    -   x) a heavy chain antigen-binding region that comprises an H-CDR1        comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and        an H-CDR3 comprising SEQ ID NO: 16 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:18, or    -   xi) a heavy chain antigen-binding region that comprises an        H-CDR1 comprising SEQ ID NO: 21, an H-CDR2 comprising SEQ ID NO:        8, and an H-CDR3 comprising SEQ ID NO: 13 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:18, or    -   xii) a heavy chain antigen-binding region that comprises an        H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO:        22, and an H-CDR3 comprising SEQ ID NO: 13 and a light chain        antigen-binding region that comprises an L-CDR1 comprising SEQ        ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3        comprising SEQ ID NO:18.

In particular embodiments, the isolated antibodies or antigen-bindingfragments according to the present invention comprise at least one, atleast two, at least three or at least four or five of the heavy chainvariable domain framework and CDR residues selected from the groupconsisting of 30S, 31S, 53S, 56S, 97K. These amino acid positionscorrespond to the amino acid positions of reference heavy chain variabledomain of SEQ ID NO: 32 and include framework and H-CDR1 and H-CDR2amino acid residues.

In particular embodiments, the isolated antibodies or antigen-bindingfragments according to the present invention comprise:

-   -   i) a variable heavy chain domain comprising SEQ ID NO: 32 and a        variable light chain domain comprising SEQ ID NO: 38; or    -   ii) a variable heavy chain domain comprising SEQ ID NO: 23 and a        variable light chain domain comprising SEQ ID NO: 37; or    -   iii) a variable heavy chain domain comprising SEQ ID NO: 24 and        a variable light chain domain comprising SEQ ID NO: 38; or    -   iv) a variable heavy chain domain comprising SEQ ID NO: 25 and a        variable light chain domain comprising SEQ ID NO: 38; or    -   v) a variable heavy chain domain comprising SEQ ID NO: 26 and a        variable light chain domain comprising SEQ ID NO: 38; or    -   vi) a variable heavy chain domain comprising SEQ ID NO: 27 and a        variable light chain domain comprising SEQ ID NO: 39; or    -   vii) a variable heavy chain domain comprising SEQ ID NO: 27 and        a variable light chain domain comprising SEQ ID NO: 38; or    -   viii) a variable heavy chain domain comprising SEQ ID NO: 28 and        a variable light chain domain comprising SEQ ID NO: 38; or    -   ix) a variable heavy chain domain comprising SEQ ID NO: 28 and a        variable light chain domain comprising SEQ ID NO: 40; or    -   x) a variable heavy chain domain comprising SEQ ID NO: 29 and a        variable light chain domain comprising SEQ ID NO: 38; or    -   xi) a variable heavy chain domain comprising SEQ ID NO: 30 and a        variable light chain domain comprising SEQ ID NO: 38; or    -   xii) a variable heavy chain domain comprising SEQ ID NO: 31 and        a variable light chain domain comprising SEQ ID NO: 38; or    -   xiii) a variable heavy chain domain comprising SEQ ID NO: 33 and        a variable light chain domain comprising SEQ ID NO: 38; or    -   xiv) a variable heavy chain domain comprising SEQ ID NO: 34 and        a variable light chain domain comprising SEQ ID NO: 38; or    -   xv) a variable heavy chain domain comprising SEQ ID NO: 35 and a        variable light chain domain comprising SEQ ID NO: 38; or    -   xvi) a variable heavy chain domain comprising SEQ ID NO: 36 and        a variable light chain domain comprising SEQ ID NO: 38.

In particular embodiments, the isolated antibodies according to thepresent invention are IgG antibody. In particular such embodiments, theisolated antibodies according to the present invention are an IgG1,IgG2, IgG3 or an IgG4 antibody. Most particularly, the isolatedantibodies according to the present invention is an IgG1 or an IgG4antibody.

In particular embodiments, the isolated antibodies according to thepresent invention comprise:

-   -   i) a heavy chain comprising SEQ ID NO: 55 and a light chain        comprising SEQ ID NO: 57; or    -   ii) a heavy chain comprising SEQ ID NO: 41 and a light chain        comprising SEQ ID NO: 56; or    -   iii) a heavy chain comprising SEQ ID NO: 42 and a light chain        comprising SEQ ID NO: 57; or    -   iv) a heavy chain comprising SEQ ID NO: 43 and a light chain        comprising SEQ ID NO: 57; or    -   v) a heavy chain comprising SEQ ID NO: 44 and a light chain        comprising SEQ ID NO: 57; or    -   vi) a heavy chain comprising SEQ ID NO: 45 and a light chain        comprising SEQ ID NO: 58; or    -   vii) a heavy chain comprising SEQ ID NO: 45 and a light chain        comprising SEQ ID NO: 57; or    -   viii) a heavy chain comprising SEQ ID NO: 46 and a light chain        comprising SEQ ID NO: 57; or    -   ix) a heavy chain comprising SEQ ID NO: 46 and a light chain        comprising SEQ ID NO: 59; or    -   x) a heavy chain comprising SEQ ID NO: 47 and a light chain        comprising SEQ ID NO: 57; or    -   xi) a heavy chain comprising SEQ ID NO: 48 and a light chain        comprising SEQ ID NO: 57; or    -   xii) a heavy chain comprising SEQ ID NO: 49 and a light chain        comprising SEQ ID NO: 57; or    -   xiii) a heavy chain comprising SEQ ID NO: 50 and a light chain        comprising SEQ ID NO: 57; or    -   xiv) a heavy chain comprising SEQ ID NO: 51 and a light chain        comprising SEQ ID NO: 57; or    -   xv) a heavy chain comprising SEQ ID NO: 52 and a light chain        comprising SEQ ID NO: 57; or    -   xvi) a heavy chain comprising SEQ ID NO: 53 and a light chain        comprising SEQ ID NO: 57; or    -   xvii) a heavy chain comprising SEQ ID NO: 54 and a light chain        comprising SEQ ID NO: 57.

In particular embodiments, the antigen-binding fragments according tothe present invention are scFv, Fab, Fab′ fragment or a F(ab′)2fragments.

In particular embodiments, the isolated antibodies or antigen-bindingfragments according to the present invention are monoclonal antibodiesor antigen-binding fragments.

In particular embodiments, the isolated antibodies or antigen-bindingfragments according to the present invention are human, humanized orchimeric antibodies or antigen-binding fragments, more particularlyfully human antibodies or antigen-binding fragments.

In particular embodiments, the isolated antibodies or antigen-bindingfragments according to the present invention are monospecificantibodies. In particular other embodiments, the isolated antibodies orantigen-binding fragments according to the present invention aremultispecific antibodies that bind to A2AP and at least one furtherantigen, such bispecific, trispecific or tetraspecific antibodies.

In a further aspect, the present invention relates to isolatedantibodies or antigen-binding fragments thereof that compete with theisolated antibodies or antigen-binding fragments according to thepresent invention for binding to A2AP.

Amino acid sequences of preferred antibodies according to the presentinvention are listed in Table 1.

TABLE 1 Amino acid sequences of preferred antibodies according to thepresent invention SEQ SEQ ID ID NO: NO: SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQIgG IgG Antibody/ ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: IDNO: Heavy Light Isotype VH H-CDR1 H-CDR2 H-CDR3 VL L-CDR1 L-CDR2 L-CDR3Chain Chain TPP-12387 23 6 7 11 37 9 10 17 41 56 hIgG1 TPP-14293 24 6 711 38 9 10 18 42 57 hIgG1 TPP-14298 25 6 7 11 38 9 10 18 43 57 hIgG1TPP-14303 26 6 7 12 38 9 10 18 44 57 hIgG1 TPP-14305 27 6 7 13 39 9 1019 45 58 hIgG1 TPP-14308 27 6 7 13 38 9 10 18 45 57 hIgG1 TPP-14313 28 67 14 38 9 10 18 46 57 hIgG1 TPP-14314 28 6 7 14 40 9 10 20 46 59 hIgG1TPP-14318 29 6 7 15 38 9 10 18 47 57 hIgG1 TPP-14323 30 6 7 16 38 9 1018 48 57 hIgG1 TPP-17041 31 6 7 13 38 9 10 18 49 57 hIgG1 TPP-17044 32 68 13 38 9 10 18 50 57 hIgG1 TPP-17045 33 6 7 13 38 9 10 18 51 57 hIgG1TPP-17048 34 6 8 13 38 9 10 18 52 57 hIgG1 TPP-17051 35 21 8 13 38 9 1018 53 57 hIgG1 TPP-17053 36 6 22 13 38 9 10 18 54 57 hIgG1 TPP-17928 326 8 13 38 9 10 18 55 57 hIgG4

Nucleic acid sequences of preferred antibodies according to the presentinvention are listed in Table 2.

TABLE 2 Nucleic acid sequences of preferred antibodies according to thepresent invention SEQ SEQ ID ID NO: NO: SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQIgG IgG Antibody/ ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: IDNO: Heavy Light Isotype VH H-CDR1 H-CDR2 H-CDR3 VL L-CDR1 L-CDR2 L-CDR3Chain Chain TPP-12387 80 63 64 68 94 66 67 74 98 113 hIgG1 TPP-14293 8163 64 68 95 66 67 75 99 114 hIgG1 TPP-14298 82 63 64 68 95 66 67 75 100114 hIgG1 TPP-14303 83 63 64 69 95 66 67 75 101 114 hIgG1 TPP-14305 8463 64 70 96 66 67 76 102 115 hIgG1 TPP-14308 84 63 64 70 95 66 67 75 102114 hIgG1 TPP-14313 85 63 64 71 95 66 67 75 103 114 hIgG1 TPP-14314 8563 64 71 97 66 67 77 103 116 hIgG1 TPP-14318 86 63 64 72 95 66 67 75 104114 hIgG1 TPP-14323 87 63 64 73 95 66 67 75 105 114 hIgG1 TPP-17041 8863 64 117 95 66 67 75 106 114 hIgG1 TPP-17044 89 63 65 70 95 66 67 75107 114 hIgG1 TPP-17045 90 63 64 70 95 66 67 75 108 114 hIgG1 TPP-1704891 63 65 70 95 66 67 75 109 114 hIgG1 TPP-17051 92 78 65 70 95 66 67 75110 114 hIgG1 TPP-17053 93 63 79 70 95 66 67 75 111 114 hIgG1 TPP-1792889 63 65 70 95 66 67 75 112 114 hIgG4

Peptide Variants

Antibodies or antigen-binding fragments of the invention are not limitedto the specific peptide sequences provided herein. Rather, the inventionalso embodies variants of these polypeptides. With reference to theinstant disclosure and conventionally available technologies andreferences, the skilled worker will be able to prepare, test and utilizefunctional variants of the antibodies disclosed herein, whileappreciating these variants having the ability to bind to A2AP fallwithin the scope of the present invention.

A variant can include, for example, an antibody that has at least onealtered complementary determining region (CDR) (hyper-variable) and/orframework (FR) (variable) domain/position, vis-à-vis a peptide sequencedisclosed herein.

By altering one or more amino acid residues in a CDR or FR region, theskilled worker routinely can generate mutated or diversified antibodysequences, which can be screened against the antigen, for new orimproved properties, for example.

A further preferred embodiment of the invention is an antibody orantigen-binding fragment in which the VH and VL sequences are selectedas shown in Table 1. The skilled worker can use the data in Table 1 todesign peptide variants that are within the scope of the presentinvention. It is preferred that variants are constructed by changingamino acids within one or more CDR regions; a variant might also haveone or more altered framework regions. Alterations also may be made inthe framework regions. For example, a peptide FR domain might be alteredwhere there is a deviation in a residue compared to a germline sequence.

Alternatively, the skilled worker could make the same analysis bycomparing the amino acid sequences disclosed herein to known sequencesof the same class of such antibodies, using, for example, the proceduredescribed by Knappik A., et al., JMB 2000, 296:57-86.

Furthermore, variants may be obtained by using one antibody as startingpoint for further optimization by diversifying one or more amino acidresidues in the antibody, preferably amino acid residues in one or moreCDRs, and by screening the resulting collection of antibody variants forvariants with improved properties. Particularly preferred isdiversification of one or more amino acid residues in CDR3 of VL and/orVH. Diversification can be done e.g. by synthesizing a collection of DNAmolecules using trinucleotide mutagenesis (TRIM) technology (Virnekas B.et al., Nucl. Acids Res. 1994, 22: 5600.). Antibodies or antigen-bindingfragments thereof include molecules with modifications/variationsincluding but not limited to e.g. modifications leading to alteredhalf-life (e.g. modification of the Fc part or attachment of furthermolecules such as PEG), altered binding affinity or altered ADCC or CDCactivity.

Conservative Amino Acid Variants

Polypeptide variants may be made that conserve the overall molecularstructure of an antibody peptide sequence described herein. Given theproperties of the individual amino acids, some rational substitutionswill be recognized by the skilled worker. Amino acid substitutions,i.e., “conservative substitutions,” may be made, for instance, on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.

For example, (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophane, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt α-helices. Similarly, certainamino acids, such as alanine, cysteine, leucine, methionine, glutamicacid, glutamine, histidine and lysine are more commonly found inα-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophanand threonine are more commonly found in (3-pleated sheets. Glycine,serine, aspartic acid, asparagine, and proline are commonly found inturns. Some preferred substitutions may be made among the followinggroups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given theknown genetic code, and recombinant and synthetic DNA techniques, theskilled scientist readily can construct DNAs encoding the conservativeamino acid variants.

Glycosylation Variants

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 using Kabat EU numbering ofthe CH2 domain of the Fc region; see, e.g., Wright et al. TrendsBiotechnol. 15: 26-32 (1997).

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the expression system (e.g. hostcell) and/or by altering the amino acid sequence such that one or moreglycosylation sites is created or removed.

In one embodiment of this invention, aglycosyl antibodies havingdecreased effector function or antibody derivatives are prepared byexpression in a prokaryotic host. Suitable prokaryotic hosts for includebut are not limited to E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

In one embodiment, antibody variants are provided having decreasedeffector function, which are characterized by a modification at theconserved N-linked site in the CH2 domains of the Fc portion of saidantibody. In one embodiment of present invention, the modificationcomprises a mutation at the heavy chain glycosylation site to preventglycosylation at the site. Thus, in one preferred embodiment of thisinvention, the aglycosyl antibodies or antibody derivatives are preparedby mutation of the heavy chain glycosylation site, —i.e., mutation ofN297 using Kabat EU numbering and expressed in an appropriate host cell.

In another embodiment of the present invention, aglycosyl antibodies orantibody derivatives have decreased effector function, wherein themodification at the conserved N-linked site in the CH2 domains of the Fcportion of said antibody or antibody derivative comprises the removal ofthe CH2 domain glycans, —i.e., deglycosylation. These aglycosylantibodies may be generated by conventional methods and thendeglycosylated enzymatically. Methods for enzymatic deglycosylation ofantibodies are well known in the art (e.g. Winkelhake & Nicolson (1976),J Biol Chem. 251(4):1074-80).

In another embodiment of this invention, deglycosylation may be achievedusing the glycosylation inhibitor tunicamycin (Nose & Wigzell (1983),Proc Natl Acad Sci USA, 80(21):6632-6). That is, the modification is theprevention of glycosylation at the conserved N-linked site in the CH2domains of the Fc portion of said antibody.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.

Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include Okazaki et al. J Mol. Biol.336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004).

Examples of cell lines capable of producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); and WO 2004/056312), andknockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8,knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688(2006)).

Antibody variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546.

Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO1997/30087; WO1998/58964; and WO1999/22764.

Fc Region Variants

In certain embodiments, one or more amino acid modifications (e.g. asubstitution) may be introduced into the Fc region of an antibody (e.g.,a human IgG1, IgG2, IgG3 or IgG4 Fc region) provided herein, therebygenerating an Fc region variant.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity) butretains FcRn binding ability. In some embodiments, alterations are madein the Fc region that result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC).

In certain embodiments, the invention contemplates an antibody variantthat possesses an increased or decreased half-live. Antibodies withincreased half-lives and improved binding to the neonatal Fc receptor(FcRn), which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J Immunol. 117:587 (1976) and Kim et al., JImmunol. 24:249 (1994)), are described in US2005/0014934 (Hinton etal.). Those antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.

In a further aspect, the present invention relates to antibodyconjugates, comprising the isolated antibodies or antigen bindingfragments according to the present invention.

Antibody Generation

An antibody of the invention may be derived from a recombinant antibodylibrary that is based on amino acid sequences that have been isolatedfrom the antibodies of a large number of healthy volunteers e.g. usingthe n-CoDeR® technology the fully human CDRs are recombined into newantibody molecules (Carlson & Söderlind, Expert Rev Mol Diagn. 2001 May;1(1):102-8). Or alternatively for example antibody libraries as thefully human antibody phage display library described in Hoet R M et al.,Nat Biotechnol 2005; 23(3):344-8) can be used to isolate A2AP-specificantibodies. Antibodies or antibody fragments isolated from humanantibody libraries are considered human antibodies or human antibodyfragments herein.

Human antibodies may be further prepared by administering an immunogento a transgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. For example,immunization of genetically engineered mice inter alia immunization ofhMAb mice (e.g. VelocImmune Mouse® or XENOMOUSE®) may be performed.

Further antibodies may be generated using the hybridoma technology (forexample see Kohler and Milstein Nature. 1975 Aug. 7; 256(5517):495-7),resulting in for example murine, rat, or rabbit antibodies which can beconverted into chimeric or humanized antibodies. Humanized antibodiesand methods of making them are reviewed, e.g., in Almagro and Fransson,Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., inRiechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. NatlAcad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34(2005) (describing specificity determining region (SDR) grafting);Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”);Dall'Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”);and Osboum et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.Cancer, 83:252-260 (2000) (describing the “guided selection” approach toFR shuffling).

Examples are provided for the generation of antibodies using arecombinant antibody library.

DNA Molecules According to the Present Invention

The present invention also relates to an isolated nucleic acid sequencethat encodes the antibody or antigen-binding fragment according to thepresent invention. The isolated nucleic acid sequence encoding theantibody or antigen-binding fragment according to the present inventioncan for instance be produced by techniques described in Sambrook et al.,1989, and Ausubel et al., 1989, or alternatively, by chemicallysynthesis. (e.g. techniques described in Oligonucleotide Synthesis(1984, Gait, ed., IRL Press, Oxford)). The DNA sequences used for theantibodies expressed are given in Table 2. These sequences are optimizedin certain cases for mammalian expression. DNA molecules of theinvention are not limited to the sequences disclosed herein, but alsoinclude variants thereof. DNA variants within the invention may bedescribed by reference to their physical properties in hybridization.The skilled worker will recognize that DNA can be used to identify itscomplement and, since DNA is double stranded, its equivalent or homolog,using nucleic acid hybridization techniques. It also will be recognizedthat hybridization can occur with less than 100% complementarity.However, given appropriate choice of conditions, hybridizationtechniques can be used to differentiate among DNA sequences based ontheir structural relatedness to a particular probe. For guidanceregarding such conditions see, Sambrook et al., 1989 supra and Ausubelet al., 1995 (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). Current Protocolsin Molecular Biology. New York: John Wiley and Sons).

Structural similarity between two polynucleotide sequences can beexpressed as a function of “stringency” of the conditions under whichthe two sequences will hybridize with one another. As used herein, theterm “stringency” refers to the extent that the conditions disfavorhybridization. Stringent conditions strongly disfavor hybridization, andonly the most structurally related molecules will hybridize to oneanother under such conditions. Conversely, non-stringent conditionsfavor hybridization of molecules displaying a lesser degree ofstructural relatedness. Hybridization stringency, therefore, directlycorrelates with the structural relationships of two nucleic acidsequences.

Hybridization stringency is a function of many factors, includingoverall DNA concentration, ionic strength, temperature, probe size andthe presence of agents which disrupt hydrogen bonding. Factors promotinghybridization include high DNA concentrations, high ionic strengths, lowtemperatures, longer probe size and the absence of agents that disrupthydrogen bonding. Hybridization typically is performed in two phases:the “binding” phase and the “washing” phase.

Functionally Equivalent DNA Variants

Yet another class of DNA variants within the scope of the invention maybe described with reference to the product they encode. Thesefunctionally equivalent polynucleotides are characterized by the factthat they encode the same peptide sequences due to the degeneracy of thegenetic code.

It is recognized that variants of DNA molecules provided herein can beconstructed in several different ways. For example, they may beconstructed as completely synthetic DNAs. Methods of efficientlysynthesizing oligonucleotides are widely available. See Ausubel et al.,section 2.11, Supplement 21 (1993). Overlapping oligonucleotides may besynthesized and assembled in a fashion first reported by Khorana et al.,J. Mol. Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section8.2. Synthetic DNAs preferably are designed with convenient restrictionsites engineered at the 5′ and 3′ ends of the gene to facilitate cloninginto an appropriate vector.

As indicated, a method of generating variants is to start with one ofthe DNAs disclosed herein and then to conduct site-directed mutagenesis.See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typicalmethod, a target DNA is cloned into a single-stranded DNA bacteriophagevehicle. Single-stranded DNA is isolated and hybridized with anoligonucleotide containing the desired nucleotide alteration(s). Thecomplementary strand is synthesized and the double stranded phage isintroduced into a host. Some of the resulting progeny will contain thedesired mutant, which can be confirmed using DNA sequencing. Inaddition, various methods are available that increase the probabilitythat the progeny phage will be the desired mutant. These methods arewell known to those in the field and kits are commercially available forgenerating such mutants.

Recombinant DNA Constructs and Expression

The present invention further provides recombinant DNA constructscomprising one or more of the nucleotide sequences according to thepresent invention. The recombinant constructs of the present inventioncan be used in connection with a vector, such as a plasmid, phagemid,phage or viral vector, into which a DNA molecule encoding an antibody ofthe invention or antigen-binding fragment thereof or variant thereof isinserted.

Thus, in one aspect, the present invention relates to a vectorcomprising a nucleic acid sequence according to the present invention.

An antibody, antigen binding portion, or variant thereof provided hereincan be prepared by recombinant expression of nucleic acid sequencesencoding light and heavy chains or portions thereof in a host cell. Toexpress an antibody, antigen binding portion, or variant thereofrecombinantly a host cell can be transfected with one or morerecombinant expression vectors carrying DNA fragments encoding the lightand/or heavy chains or portions thereof such that the light and heavychains are expressed in the host cell. Standard recombinant DNAmethodologies are used to prepare and/or obtain nucleic acids encodingthe heavy and light chains, incorporate these nucleic acids intorecombinant expression vectors and introduce the vectors into hostcells, such as those described in Sambrook, Fritsch and Maniatis (eds.),Molecular Cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.No. 4,816,397 by Boss et al.

In addition, the nucleic acid sequences encoding variable regions of theheavy and/or light chains can be converted, for example, to nucleic acidsequences encoding full-length antibody chains, Fab fragments, or toscFv. The VL- or VH-encoding DNA fragment can be operatively linked,(such that the amino acid sequences encoded by the two DNA fragments arein-frame) to another DNA fragment encoding, for example, an antibodyconstant region or a flexible linker. The sequences of human heavy chainand light chain constant regions are known in the art (see e.g., Kabat,E. A., el al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification.

To create a polynucleotide sequence that encodes a scFv, the VH- andVL-encoding nucleic acids can be operatively linked to another fragmentencoding a flexible linker such that the VH and VL sequences can beexpressed as a contiguous single-chain protein, with the VL and VHregions joined by the flexible linker (see e.g., Bird et al. (1988)Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USAMcCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, antigen binding fragments thereof or variantsthereof standard recombinant DNA expression methods can be used (see,for example, Goeddel; Gene Expression Technology. Methods in Enzymology185, Academic Press, San Diego, Calif. (1990)). For example, DNAencoding the desired polypeptide can be inserted into an expressionvector which is then transfected into a suitable host cell. Suitablehost cells are prokaryotic and eukaryotic cells. Examples forprokaryotic host cells are e.g. bacteria, examples for eukaryotic hostscells are yeasts, insects and insect cells, plants and plant cells,transgenic animals, or mammalian cells. Introduction of the recombinantconstruct into the host cell can be carried out using standardtechniques such as calcium phosphate transfection, DEAE dextran mediatedtransfection, electroporation, transduction or phage infection.

In some embodiments, the DNAs encoding the heavy and light chains areinserted into separate vectors. In other embodiments, the DNA encodingthe heavy and light chains is inserted into the same vector. It isunderstood that the design of the expression vector, including theselection of regulatory sequences is affected by factors such as thechoice of the host cell, the level of expression of protein desired andwhether expression is constitutive or inducible.

Thus, in a further aspect, the present invention relates to an isolatedcell expressing the antibody or antigen-binding fragment according tothe present invention and/or comprising the nucleic acid according tothe present invention or the vector according to the present invention.

The isolated cell can be virtually any cell for which expression vectorsare available. The isolated cell can for example a higher eukaryotichost cell, such as a mammalian cell, a lower eukaryotic host cell, suchas a yeast cell, and may be a prokaryotic cell, such as a bacterialcell.

In a further aspect, the present invention relates to a method ofproducing the isolated antibody or antigen-binding fragment according tothe present invention comprising culturing of the cell according to thepresent invention. In particular embodiments, the cell according to thepresent invention is cultivated under suitable conditions for antibodyexpression and the antibody or antigen-binding fragment is recovered. Inparticular embodiments, the antibody or antigen-binding fragment ispurified, particularly to at least 95% homogeneity by weight.

Bacterial Expression

Useful expression vectors for bacterial use are constructed by insertinga DNA sequence encoding a desired protein together with suitabletranslation initiation and termination signals in operable reading phasewith a functional promoter. The vector will comprise one or morephenotypic selectable markers and an origin of replication to ensuremaintenance of the vector and, if desirable, to provide amplificationwithin the host. Suitable prokaryotic hosts for transformation includebut are not limited to E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

Bacterial vectors may be, for example, bacteriophage-, plasmid- orphagemid-based. These vectors can contain a selectable marker and abacterial origin of replication derived from commercially availableplasmids typically containing elements of the well-known cloning vectorpBR322 (ATCC 37017). Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter is de-repressed/induced by appropriate means (e.g.,temperature shift or chemical induction) and cells are cultured for anadditional period. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the proteinbeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of antibodies or to screen peptidelibraries, for example, vectors which direct the expression of highlevels of fusion protein products that are readily purified may bedesirable.

Therefore, an embodiment of the present invention is an expressionvector comprising a nucleic acid sequence encoding for the novelantibodies of the present invention.

Antibodies of the present invention or antigen-binding fragments thereofor variants thereof include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic host, including, for example, E. coli,Bacillus subtilis, Salmonella typhimurium and various species within thegenera Pseudomonas, Streptomyces, and Staphylococcus, preferably, fromE. coli cells.

Mammalian Expression

Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. Expression of theantibodies may be constitutive or regulated (e.g. inducible by additionor removal of small molecule inductors such as Tetracyclin inconjunction with Tet system). For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. No.5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S.Pat. No. 4,968,615 by Schaffner et al. The recombinant expressionvectors can also include origins of replication and selectable markers(see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). Suitableselectable markers include genes that confer resistance to drugs such asG418, puromycin, hygromycin, blasticidin, zeocin/bleomycin ormethotrexate or selectable marker that exploit auxotrophies such asGlutamine Synthetase (Bebbington et al., Biotechnology (N Y). 1992February; 10(2):169-75), on a host cell into which the vector has beenintroduced. For example, the dihydrofolate reductase (DHFR) gene confersresistance to methotrexate, neo gene confers resistance to G418, the bsdgene from Aspergillus terreus confers resistance to blasticidin,puromycin N-acetyl-transferase confers resistance to puromycin, the Shble gene product confers resistance to zeocin, and resistance tohygromycin is conferred by the E. coli hygromycin resistance gene (hygor hph). Selectable markers like DHFR or Glutamine Synthetase are alsouseful for amplification techniques in conjunction with MTX and MSX.

Transfection of the expression vector into a host cell can be carriedout using standard techniques such as electroporation, nucleofection,calcium-phosphate precipitation, lipofection, polycation-basedtransfection such as polyethlylenimine (PEI)-based transfection andDEAE-dextran transfection.

Suitable mammalian host cells for expressing the antibodies, antigenbinding fragments thereof or variants thereof provided herein includeChinese Hamster Ovary (CHO cells) such as CHO-K1, CHO-S, CHO-K1SV[including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 June;33(2):405-12, used with a DHFR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621; and otherknockout cells exemplified in Fan et al., Biotechnol Bioeng. 2012 April;109(4):1007-15], NS0 myeloma cells, COS cells, HEK293 cells, HKB11cells, BHK21 cells, CAP cells, EB66 cells, and SP2 cells.

Expression might also be transient or semi-stable in expression systemssuch as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E,HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S,CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7 or CAP-T cells (forinstance Durocher et al., Nucleic Acids Res. 2002 Jan. 15; 30(2):E9).

In some embodiments, the expression vector is designed such that theexpressed protein is secreted into the culture medium in which the hostcells are grown. The antibodies, antigen binding fragments thereof orvariants thereof can be recovered from the culture medium using standardprotein purification methods.

Purification

Antibodies of the invention or antigen-binding fragments thereof orvariants thereof can be recovered and purified from recombinant cellcultures by well-known methods including, but not limited to ammoniumsulfate or ethanol precipitation, acid extraction, Protein Achromatography, Protein G chromatography, anion or cation exchangechromatography, phospho-cellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. High performance liquidchromatography (“HPLC”) can also be employed for purification. See,e.g., Colligan, Current Protocols in Immunology, or Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g.,Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein byreference.

Antibodies of the present invention or antigen-binding fragments thereofor variants thereof include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a eukaryotic host, including, for example, yeast, higherplant, insect and mammalian cells. Depending upon the host employed in arecombinant production procedure, the antibody of the present inventioncan be glycosylated or can be non-glycosylated. Such methods aredescribed in many standard laboratory manuals, such as Sambrook, supra,Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and20.

In preferred embodiments, the antibody is purified (1) to greater than95% by weight of antibody as determined e.g. by the Lowry method, UV-Visspectroscopy or by SDS-Capillary Gel electrophoresis (for example on aCaliper LabChip GXII, GX 90 or Biorad Bioanalyzer device), and infurther preferred embodiments more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing ornon-reducing conditions using Coomassie blue or, preferably, silverstain. Isolated naturally occurring antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

Therapeutic Methods

Therapeutic methods involve administering to a subject in need oftreatment a therapeutically effective amount of an antibody or anantigen-binding fragment thereof or a variant thereof contemplated bythe invention. A “therapeutically effective” amount hereby is defined asthe amount of an antibody or antigen-binding fragment that is ofsufficient quantity to increase plasmin mediated clot lysis in asubject—either as a single dose or according to a multiple dose regimen,alone or in combination with other agents, which leads to thealleviation of an adverse condition, yet which amount is toxicologicallytolerable. The subject may be a human or non-human animal (e.g., rabbit,rat, mouse, dog, monkey or other lower-order primate).

Thus, in one aspect, the present invention relates to the isolatedantibody or antigen-binding fragment according the present invention orto a conjugate comprising the isolated antibody or antigen-bindingfragment according the present invention or to a pharmaceuticalcomposition comprising the isolated antibody or antigen-binding fragmentaccording the present invention for use in the treatment or prophylaxisof diseases.

The isolated antibodies or antigen-binding fragments according to thepresent invention can be used as a therapeutic or a diagnostic tool in avariety of A2AP associated disorders and/or diseases associated withischemic events due to partial or complete vessel occlusion.

An ischemic event may be due to the partial or complete occlusion of onevessel but it may be also the result of a partial or complete occlusionof more than one vessel whereby some vessels may be partially occludedand some vessels may be complete occluded.

Thus, in a further aspect, the present invention relates to the isolatedantibodies or antigen-binding fragments according the present inventionor to conjugates comprising the isolated antibodies or antigen-bindingfragments according the present invention or to pharmaceuticalcompositions comprising the isolated antibodies or antigen-bindingfragments according the present invention for use in the treatment orprophylaxis of disorders or diseases associated with ischemic events dueto partial or complete vessel occlusion, such as ischemic stroke, acutecoronary syndrome, peripheral artery disease, myocardial infarction,deep vein thrombosis, pulmonary embolism, venous thrombosis, or shuntthrombosis.

In a further aspect, the present invention relates to the isolatedantibodies or antigen-binding fragments according the present inventionor to conjugates comprising the isolated antibodies or antigen-bindingfragments according the present invention or to pharmaceuticalcompositions comprising the isolated antibodies or antigen-bindingfragments according the present invention for treatment or prophylaxisof diseases, in particular of disorders or diseases associated withischemic events due to partial or complete vessel occlusion, such asischemic stroke, acute coronary syndrome, peripheral artery disease,myocardial infarction, deep vein thrombosis, pulmonary embolism, venousthrombosis, or shunt thrombosis.

In a further aspect, the present invention relates to the use ofisolated antibodies or antigen-binding fragments according the presentinvention or to conjugates comprising the isolated antibodies orantigen-binding fragments according the present invention or topharmaceutical compositions comprising the isolated antibodies orantigen-binding fragments according the present invention in a method oftreatment or prophylaxis of diseases, in particular of disorders ordiseases associated with ischemic events due to partial or completevessel occlusion, such as ischemic stroke, acute coronary syndrome,peripheral artery disease, myocardial infarction, deep vein thrombosis,pulmonary embolism, venous thrombosis, or shunt thrombosis.

In a further aspect, the present invention relates to use of isolatedantibodies or antigen-binding fragments according the present inventionor to conjugates comprising the isolated antibodies or antigen-bindingfragments according the present invention or to pharmaceuticalcompositions comprising the isolated antibodies or antigen-bindingfragments according the present invention for the preparation of apharmaceutical composition, preferably a medicament, for the prophylaxisor treatment of diseases, in particular of disorders or diseasesassociated with ischemic events due to partial or complete vesselocclusion, such as ischemic stroke, acute coronary syndrome, peripheralartery disease, myocardial infarction, deep vein thrombosis, pulmonaryembolism, venous thrombosis, or shunt thrombosis.

In a further aspect, the present invention relates to methods oftreatment or prophylaxis of diseases, in particular of disorders ordiseases associated with ischemic events due to partial or completevessel occlusion, such as ischemic stroke, acute coronary syndrome,peripheral artery disease, myocardial infarction, deep vein thrombosis,pulmonary embolism, venous thrombosis, or shunt thrombosis, using aneffective amount of an isolated antibody or antigen-binding fragmentaccording the present invention or to a conjugate comprising theisolated antibody or antigen-binding fragment according the presentinvention or to a pharmaceutical composition comprising the isolatedantibody or antigen-binding fragment according the present invention.The disorders mentioned above have been well characterized in humans,but also exist with a similar etiology in other animals, includingmammals, and can be treated by administering pharmaceutical compositionsaccording to the present invention.

The antibodies or the antigen-binding fragments according to the presentinvention or variants thereof might be co-administered with knownmedications, and in some instances the antibody or antigen-bindingfragment thereof might itself be modified. For example, an antibody oran antigen-binding fragment thereof or a variant thereof could beconjugated to a drug or to another peptide or protein to potentiallyfurther increase efficacy.

Antibodies of the present invention or antigen-binding fragments thereofor variants thereof may be administered as the sole pharmaceutical agentor in combination with one or more additional therapeutic agents wherethe combination causes no unacceptable adverse effects.

Thus, in a further aspect, the present invention relates to the isolatedantibodies or antigen-binding fragments according to the presentinvention or the conjugates according to the present invention or thepharmaceutical compositions according to the present invention for usein simultaneous, separate, or sequential combination with one or morefurther therapeutically active compounds.

Non-limiting examples of therapeutically active compounds to be used incombination with the antibodies or antigen-binding fragments accordingto the present invention are:

-   -   i) inhibitors of the coagulation cascade like plasminogen        activators (thrombolytics/fibrinolytics) as well as compounds        increasing thrombolysis and/or fibrinolysis (like tissue        plasminogen activator (t-PA), streptokinase, reteplase, and        urokinase) or inhibitors of the plasminogen activator inhibitor        (PAI) or thrombin-activatable fibrinolysis inhibitors (TAFI);    -   ii) anticoagulants like non-fractionated heparins, low molecular        weight heparins, heparinoid, hirudin, bivalirudin and/or        argatroban; direct oral anticoagulants/non-vitamin K        anticoagulants like Factor Xa inhibitors, e.g. apixaban,        edoxaban, and rivaroxaban, and Thrombin inhibitors, e.g.        dabigatran.    -   iii) platelet aggregation inhibitors, like aspirin, clopidogrel,        cilostazol, prasugrel, ticagrelor, cangrelor, and others.

Combination therapy includes administration of a single pharmaceuticaldosage formulation which comprises an antibody or antigen-bindingfragment according to the present invention or a variant thereof and oneor more additional therapeutic agents, as well as administration of anantibody or antigen-binding fragment according to the present inventionand each additional therapeutic agent in its own separate pharmaceuticaldosage formulation. For example, an antibody of the invention or anantigen-binding fragment thereof or a variant thereof and a therapeuticagent may be administered to the patient together in a single liquidcomposition, or each agent may be administered in separate dosageformulation.

Where separate dosage formulations are used, the antibody orantigen-binding fragment according to the present invention or thevariant thereof and one or more additional therapeutic agents may beadministered at essentially the same time (e.g., concurrently) or atseparately staggered times (e.g., sequentially).

The antibodies or the antigen-binding fragments according to the presentinvention or variants thereof might be used in combination with surgicalinterventions, like but not limited to mechanical embolectomy,thrombectomy, clot retrieval devices, cerebral revascularization.

Diagnostic Methods

Furthermore, the antibodies or antigen-binding fragments according tothe present invention may be utilized, as such or in compositions, inresearch and diagnostics, or as analytical reference standards, and thelike.

Anti-A2AP antibodies or antigen-binding fragments thereof can be usedfor detecting the presence of A2AP. Thus, in a further aspect, thepresent invention relates to the isolated antibodies or antigen-bindingfragments according to the present invention or the antibody conjugatesaccording to the present invention for use as a diagnostic agent.

Pharmaceutical Compositions and Administration

In a further aspect, the present invention relates to pharmaceuticalcompositions comprising the isolated antibodies or antigen-bindingfragments according to the present invention or the antibody conjugatesaccording to the present invention. To treat any of the foregoingdisorders, pharmaceutical compositions for use in accordance with thepresent invention may be formulated in any conventional manner using oneor more physiologically acceptable carriers, excipients, or auxiliaries.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences (Ed.Maack Publishing Co, Easton, Pa.).

The antibody or antigen-binding fragment according to the presentinvention can be administered by any suitable means, which can vary,depending on the type of disorder being treated. Possible administrationroutes include oral, parenteral, and topical administration. Methods ofparenteral delivery include intra-arterial, intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition, the antibodyor antigen-binding fragment according to the present invention may beadministered by pulse infusion, with, e.g., declining doses of theantibody. Preferably, administration is by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or prolonged. The amount to be administered willdepend on a variety of factors such as the clinical symptoms, weight ofthe individual, whether other drugs are administered, and the like. Theskilled artisan will recognize that the route of administration willvary depending on the disorder or condition to be treated.

The pharmaceutical composition according to the present inventioncomprises the antibody or antigen-binding fragment according to thepresent invention alone or in combination with at least one other agent,such as a stabilizing compound. The antibody or antigen-binding fragmentaccording to the present invention may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. In particular embodiments,the pharmaceutical composition according to the present invention maycomprise one or more further pharmaceutically active compounds, inparticular one or more further pharmaceutically active compounds thatare suitable to treat A2AP associated disorders and/or disordersassociated with ischemic events due to partial or complete vesselocclusion. Any of these agents can be administered to a patient alone,or in combination with other agents or drugs, in pharmaceuticalcompositions where it is mixed with excipient(s) or pharmaceuticallyacceptable carriers. In particular embodiments, the pharmaceuticallyacceptable carrier is pharmaceutically inert.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl-cellulose, hydroxypropylmethylcellulose, or sodiumcarboxymethyl cellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores can be provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.Dyestuffs or pigments may be added to the tablets or dragee coatings forproduct identification or to characterize the quantity of activecompound, i.e. dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, andoptionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances that increase viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with acids, including but not limited to hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents that are the correspondingfree base forms. In other cases, the preferred preparation may be alyophilized powder in 1 mM-50 mM histidine or phosphate or Tris, 0.1%-2%sucrose and/or 2%-7% mannitol at a pH range of 4.5 to 7.5 optionallycomprising additional substances like polysorbate that is combined withbuffer prior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in an acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of anti-A2AP antibodies orantigen-binding fragment thereof, such labeling would include amount,frequency and method of administration.

Therapeutically Effective Dose

Pharmaceutical compositions suitable for use according to the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose, e.g.,treatment of a particular disease state characterized by ischemic eventsdue to partial or complete vessel occlusion.

The determination of an effective dose is well within the capability ofthose skilled in the art. Determining a therapeutically effective amountof the novel antibody of this invention or an antigen-binding fragmentthereof or a variant thereof, largely will depend on particular patientcharacteristics, route of administration, and the nature of the disorderbeing treated. General guidance can be found, for example, in thepublications of the International Conference on Harmonization and inREMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528(18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990).More specifically, determining a therapeutically effective amount willdepend on such factors as toxicity and efficacy of the medicament.Toxicity may be determined using methods well known in the art and foundin the foregoing references. Efficacy may be determined utilizing thesame guidance in conjunction with the methods described below in theExamples.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, or in animal models, usuallymice, rabbits, dogs, pigs or monkeys. The animal model is also used toachieve a desirable concentration range and route of administration.Such information can then be used to determine useful doses and routesfor administration in humans.

A therapeutically effective dose refers to that amount of antibody orantigen-binding fragment thereof, that ameliorates the symptoms orcondition. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED₅₀/LD₅₀.Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors that may be taken into account include theseverity of the disease state, age, weight and gender of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions might be administered for example every 3 to4 days, every week, once every two weeks, or once every three weeks,depending on half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 10 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.

Kits

In a further aspect, the present invention relates to kits comprisingthe isolated antibodies or antigen-binding fragments according to thepresent invention or the conjugates according to the present inventionand instructions for use. In particular embodiments, the kits compriseone or more containers filled with one or more of the ingredients of theaforementioned compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, reflecting approval by the agency of themanufacture, use or sale of the product for human administration.

SHORT DESCRIPTION OF FIGURES

FIG. 1 : Panning strategy for finding cross-species specific,neutralizing anti-alpha2-Antiplasmin antibodies.

Four major strategies for selections on biotinylated antigens aredepicted. Where indicated, prior to each round of selection a depletionstep on a relevant or a non-alpha2-Antiplasmin biotinylated protein wasincluded.

FIG. 2 : ELISA-based analysis of the binding of Fab 431A-M080-001 tohuman alpha2-Antiplasmin and rabbit alpha2-Antiplasmin

The specific binding of Fab 431A-M080-001 to human (black columns) andrabbit (grey columns) alpha2-Antiplasmin as assessed in an ELISA assayis shown. Antigens were coated to microtiter plates at a finalconcentration of 1 μg/ml. For this, supernatants of transfected cellswere diluted in Phosphate-buffered Saline (PBS) by the factor of 1:1.5,1:4.5, 1:13.5, 1:40.5, 1:121.5, 1:364.5, 1:1093.5. Relative fluorescenceunits (RFU, ordinate) are plotted against the diluted Fabs (abscissa).For further details see Example 3.

FIG. 3 : Analyzing Fab 431A-M080-001 for function blocking activity.

The function blocking activity of Fab 431A-M080-001 as measured in thePlasmin—alpha2-Antiplasmin biochemical assay is depicted. For this,supernatants from mammalian cells containing the Fab of interest werepre-incubated with human or rabbit alpha2-Antiplasmin, followed by theaddition of human Plasmin and the fluorogenic plasmin substrate. Therelative fluorescence units resulting from cleavage of the substrate byPlasmin were measured. Resulting data are presented as percentage ofinhibition. Left light-grey column is representing the neutralization ofhuman alpha2-Antiplasmin, right dark-grey column is representing theneutralization of rabbit alpha2-Antiplasmin. See Example 4 for detaileddescription of the biochemical assay.

FIG. 4 : Binding activity of antibody TPP-12387 on human and rabbitalpha2-Antiplasmin.

According to the method described in Example 3, antibody TPP-12387 wastested for its ability to bind human and rabbit alpha2-Antiplasmin in adose-dependent manner. Binding activities towards humanalpha2-Antiplasmin are shown in the left panel, towards rabbitalpha2-Antiplasmin are shown in the right panel of this figure. Thebinding activities were calculated as EC50 in M values. One doseresponse curve is shown as example from two to three independentexperiments performed in quadruplicate: EC50 (human A2AP) was 1.2E-07 M;EC50 (rabbit A2AP) was 6.0E-09 M.

FIG. 5 : Neutralizing activity of antibody TPP-12387 on human and rabbitalpha2-Antiplasmin.

According to the method described in Example 4, antibody TPP-12387 wastested for its ability to block the activity of human and rabbitalpha2-Antiplasmin in a dose-dependent manner. Neutralizing activitiestowards human alpha2-Antiplasmin are shown in the left panel, towardsrabbit alpha2-Antiplasmin are shown in the right panel of this figure.Function blocking activities were calculated as EC50 in M values. Onedose response curve is shown as example from two to three independentexperiments performed in quadruplicate: EC50 (human A2AP) was 1.7E-07 M;EC50 (rabbit A2AP) was 1.4E-09 M.

FIG. 6 : Binding and function blocking activity of antibody TPP-12387 onCynomolgus alpha2-Antiplasmin

According to the methods described in Example 3 and Example 4, antibodyTPP-12387 was tested for its ability to block the activity of cynomolgusalpha2-Antiplasmin in a dose-dependent manner. Binding activity of theantibody towards cynomolgus alpha-2Antiplasmin is shown in FIG. 6.1 ,its neutralizing activity in FIG. 6.2 . Activities were calculated asEC50 in M values. One dose response curve is shown as example from twoto three independent experiments performed in quadruplicate: EC50(cynomolgus A2AP binding) was 9.9E-08 M; EC50 (cynomolgus A2AP activityblocking) was 1.6E-07 M.

FIG. 7 : Binding and function blocking activities of TPP-12387 variantstowards human alpha2-Antiplasmin.

According to the methods described in Example 3 and Example 4,antibodies TPP-14323 (7.17, 7.18), TPP-14318 (7.15, 7.16), TPP-14314(7.13, 7.14), TPP-14313 (7.11, 7.12), TPP-14308 (7.9, 7.10), TPP-14305(7.7, 7.8), TPP-14303 (7.5, 7.6), TPP-14298 (7.3; 7.4), and TPP-14293(7.1; 7.2) were tested for their ability to bind to and to block theactivity of human alpha2-Antiplasmin in a dose-dependent manner. Bindingactivities towards human alpha2-Antiplasmin are shown in 7.1, 7.3, 7.5,7.7, 7.9, 7.11, 7.13, 7.15, 7.17, neutralizing activities are shown in7.2, 7.4, 7.6, 7.8, 7.10, 7.12, 7.14, 7.16, 7.18. Binding and functionblocking activities were calculated as EC50 in M values (table 3.3). Foreach antibody, one dose response curve is shown as example from two tothree independent experiments performed in quadruplicate.

FIG. 8 : Testing germline variants of TPP-14308 for binding andneutralizing human alpha2-Antiplasmin.

47 antibodies resulting from the germlining approach of TPP-14308 weretested for ability to bind and to block the activity of humanalpha2-Antiplasmin in a dose-dependent manner in comparison toTPP-14308. 6 antibodies resulting from the germlining approach ofTPP-14308 (TPP-17041, TPP-17044, TPP-17045, TPP-17048, TPP-17051,TPP-17053), show improved binding activities and/or neutralizingactivities. Binding activities towards human alpha2-Antiplasmin areshown in 8.1, 8.3, 8.5, 8.7, 8.9, 8.11, neutralizing activities areshown in 8.2, 8.4, 8.6, 8.8, 8.10, 8.12. Binding and function blockingactivities were calculated as EC50 in M values (table 3.5). For eachantibody, one dose response curve is shown as example from two to threeindependent experiments performed in quadruplicate (squares=TPP 17308,circle=TPP-17041, TPP-17044, TPP-17045, TPP-17048, TPP-17051 orTPP-17053).

FIG. 9 : Neutralizing activity of TPP-17044 IgG1 antibody on humanalpha2-Antiplasmin from different species.

Testing of TPP-17044 for function blocking activity according to themethods described in Example 4 on human (9.1), cynomolgus (9.2), andrabbit (9.3) alpha2-Antiplasmin is shown. Neutralizing activity wascalculated as EC50 in M values. For this antibody, one dose responsecurve is shown as example from two to three independent experimentsperformed in quadruplicate. Function blocking activity of TPP-17044 forhuman alpha2-Antiplasmin was 4.4E-10 M (as shown in FIGS. 9.1 ), and5.4E-10 M for the second and 5.0E-10 M for the third experiment. For theinhibition of Cynomolgus alpha2-Antiplasmin values were 4.6E-10 M (FIG.9.2 ), 4.9E-10 M for the second experiment and 5.1E-10 M for the thirdexperiment. Rabbit alpha2-Antiplasmin was blocked in its activity byTPP-17044 with IC50 values of 2.7E-08 M (FIG. 9.3 ), 3.6E-08 M for asecond experiment and 2.9E-08 M for a third experiment.

FIG. 10 : Neutralizing activity of TPP-17928 IgG4 antibody on humanalpha2-Antiplasmin from different species.

Testing of TPP-17928 for function blocking activity according to themethods described in Example 4 on human (10.1), cynomolgus (10.2), andrabbit (10.3) alpha2-Antiplasmin is shown. Neutralizing activity wascalculated as EC50 in M values. For this antibody, one dose responsecurve is shown as example from two to three independent experimentsperformed in quadruplicate. Function blocking activity of TPP-17928 forhuman alpha2-Antiplasmin was 1.1E-10 M (as shown in FIGS. 10.1 ), and1.6E-10 M for the second experiment. For the inhibition of Cynomolgusalpha2-Antiplasmin values were 2.6E-10 M (FIG. 3.4E-10 M for the secondexperiment and 2.9E-10 M for the third experiment. Rabbitalpha2-Antiplasmin was blocked in its activity by TPP-17928 with IC50values of 1.5E-08 M (FIG. 10.3 ), 1.9E-10 M for a second experiment and1.6E-10 M for a third experiment.

FIG. 11 : Reduction of clot lysis time by TPP-17928.

Antibody TPP-17928 reduces the tPA-induced clot lysis time in human(triangles) and in rabbit (squares) plasma, respectively, in adose-dependent manner. Activity was calculated as IC50 in M values. Thecurves represent the mean (+/−SD) from three independent experiments.For experimental details see Example 7. IC50 (human plasma) was 2.5E-07M; EC50 (rabbit plasma) was 2.3E-07 M.

FIG. 12 : in vivo effects of TPP-17928 on clot lysis.

Animals received fluorescently-labeled plasma clots 30 min prior tomeasurement; respective treatment was administered at time point 0 min.Over 360 min plasma samples were drawn, and the amount of plasmafluorescence as indirect parameter of clot dissolution was measured.Effects of different concentrations of TPP-17928 (FIG. 12.1 , controlcircle; 3.75 mg/kg open circle; 7.5 mg/kg open square; 15 mg/kg opentriangle), and effects of different concentrations of), tPA (FIG. 12.2 ,control circle, 0.125 mg/kg triangle, 0.25 mg/kg square, 1 mg/kgdiamond) or the combination of both (FIG. 12.1 , 15 mg/kg+0.125 mg/kgtPA (open diamond) on clot lysis were measured. Relative fluorescenceunits (rFU, ordinate) are plotted against the timepoints at which plasmasamples have been taken (abscissa). Values are mean+/−SD. TPP-17928alone has a dose-dependent effect on clot dissolution. After applicationof the antibody of the invention an activity maximum is achieved ataround 60 min which then results in a lasting effect over the wholeexperimental time (360 min). Also, tPA-treatment shows a dose-dependenteffect on clot dissolution. tPA has a fast and steep increase of clotdissolution (maximum after 15 min) but does not show a longer lastingeffect as observed for TPP-17928. The co-administration of TPP-17928 toa low dose tPA leads to a faster clot dissolution than theadministration of the single compounds. For details see Example 8.

FIG. 13 : Determination of tPA and TPP-17928 induced ear bleeding time.

Simultaneously to plasma fluorescence measurement (shown in FIG. 12 ),ear bleeding time was determined at time point 0 min following compoundadministration. For each treatment group, the bleeding time in seconds(sec) is shown. Values are mean+/−SEM.

Column 1: Control

Column 2: 0.125 mg/kg tPA,

Column 3: 0.25 mg/kg tPA

Column 4: 1 mg/kg tPA

Column 5: 3.75 mg/kg TPP-17928

Column 6: 7.5 mg/kg TPP-17928

Column 7: 15 mg/kg TPP-17928

Column 8: 15 mg/kg TPP-17928+0.125 mg/kg tPA

FIG. 14 : Effect of 77A3 and antibodies of the invention on plasmin

The results of the A2AP function blocking assay as described in example10 are shown.

The following antibodies and antibody concentrations were used:

FIG. 14.1 : 6.1E-11-3.0E-08 M 77A3 (left diagram) and 6.11E-11-1.0E-06 M77A3 (right diagram)

FIG. 14.2 : 6.11E-11-1E-06 M 77A3 (circle) and 6.1E-11-1.0E-06 MTPP-17041 (triangle)

FIG. 14.3 : 6.1E-11-1.0E-06 M 77A3 (circle) and 6.1E-11-1.0E-06 MTPP-17044 (square)

FIG. 14.4 : 6.1E-11-1Ee-06 M 77A3 (circle) and 6.1E-11-1.0E-06 MTPP-17045 (triangle)

FIG. 14.5 : 6.1E-11-1.0E-06 M 77A3 (circle) and 6.1E-11-1.0E-06 MTPP-17048 (diamond)

FIG. 14.6 : 6.1E-11-1.0E-06 M 77A3 (circle) and 6.1E-11-1.0E-06 MTPP-17051 (triangle).

FIG. 14.7 : 6.1E-11-1.0E-06 M 77A3 (circle) and 6.1E-11-1.0E-06 MTPP-17053 (square).

An increase of 77A3 concentration up to 0.03 μM resulted into anincrease of fluorescence signal due to the cleavage of the fluorogenicsubstrate 1-1275 by plasmin. However, a further increase in 77A3concentration resulted in a decrease of fluorescence signal. Thatindicates that, testing antibody 77A3 in the biochemical assay describedin Example 4 up to a concentration of 0.03 μM leads to blockade ofalpha2-Antiplasmin and that a further increase in 77A3 antibodyconcentration leads to a decline in plasmin activity, resulting in acomplete inhibition of plasmin activity at a 77A3 concentration of 1 μM(FIG. 14.1 ). Surprisingly, in comparison to this finding, testingantibodies of the invention up to 1 did not result into a decrease offluorescence signal indicating that testing antibodies of the inventionhave no impact on plasmin activity (FIGS. 14.2-14.7 ).

FIG. 15 : Amino acid sequences of preferred antibodies according to thepresent invention

Amino acid sequences of VH, H-CDR1, H-CDR2, H-CDR2, H-CDR3, VL, L-CDR1,L-CDR2, L-CDR3, heavy chain and light chain of preferred antibodiesaccording to the present invention are depicted.

FIG. 16 : Effect of 77A3 and antibodies of the invention on theproteolytic activity of plasmin

The plasmin activity in dependency of the used antibody concentration asdescribed in example 11 is shown for 77A3 (squares) and TPP-17928(circle). One dose response curve from two to three independentexperiments performed in duplicates is shown as example. 77A3 shows aninhibitory effect on plasmin activity in a concentration-dependentmanner (IC50 1.7 μM) whereas TPP-17928 surprisingly does not inhibitplasmin activity up to a concentration of 10 μM (see also tabularoverview of IC50 values in example 11).

SEQUENCE LISTING

A sequence listing is enclosed which discloses the following sequences:

SEQ ID NO: Type / sequence SEQ ID NO: 1 human A2AP; AA sequenceMet Ala Leu Leu Trp Gly Leu Leu Val Leu Ser Trp Ser Cys Leu GlnGly Pro Cys Ser Val Phe Ser Pro Val Ser Ala Met Glu Pro Leu GlyArg Gln Leu Thr Ser Gly Pro Asn Gln Glu Gln Val Ser Pro Leu ThrLeu Leu Lys Leu Gly Asn Gln Glu Pro Gly Gly Gln Thr Ala Leu LysSer Pro Pro Gly Val Cys Ser Arg Asp Pro Thr Pro Glu Gln Thr HisArg Leu Ala Arg Ala Met Met Ala Phe Thr Ala Asp Leu Phe Ser LeuVal Ala Gln Thr Ser Thr Cys Pro Asn Leu Ile Leu Ser Pro Leu SerVal Ala Leu Ala Leu Ser His Leu Ala Leu Gly Ala Gln Asn His ThrLeu Gln Arg Leu Gln Gln Val Leu His Ala Gly Ser Gly Pro Cys LeuPro His Leu Leu Ser Arg Leu Cys Gln Asp Leu Gly Pro Gly Ala PheArg Leu Ala Ala Arg Met Tyr Leu Gln Lys Gly Phe Pro Ile Lys GluAsp Phe Leu Glu Gln Ser Glu Gln Leu Phe Gly Ala Lys Pro Val SerLeu Thr Gly Lys Gln Glu Asp Asp Leu Ala Asn Ile Asn Gln Trp ValLys Glu Ala Thr Glu Gly Lys Ile Gln Glu Phe Leu Ser Gly Leu ProGlu Asp Thr Val Leu Leu Leu Leu Asn Ala Ile His Phe Gln Gly PheTrp Arg Asn Lys Phe Asp Pro Ser Leu Thr Gln Arg Asp Ser Phe HisLeu Asp Glu Gln Phe Thr Val Pro Val Glu Met Met Gln Ala Arg ThrTyr Pro Leu Arg Trp Phe Leu Leu Glu Gln Pro Glu Ile Gln Val AlaHis Phe Pro Phe Lys Asn Asn Met Ser Phe Val Val Leu Val Pro ThrHis Phe Glu Trp Asn Val Ser Gln Val Leu Ala Asn Leu Ser Trp AspThr Leu His Pro Pro Leu Val Trp Glu Arg Pro Thr Lys Val Arg LeuPro Lys Leu Tyr Leu Lys His Gln Met Asp Leu Val Ala Thr Leu SerGln Leu Gly Leu Gln Glu Leu Phe Gln Ala Pro Asp Leu Arg Gly IleSer Glu Gln Ser Leu Val Val Ser Gly Val Gln His Gln Ser Thr LeuGlu Leu Ser Glu Val Gly Val Glu Ala Ala Ala Ala Thr Ser Ile AlaMet Ser Arg Met Ser Leu Ser Ser Phe Ser Val Asn Arg Pro Phe LeuPhe Phe Ile Phe Glu Asp Thr Thr Gly Leu Pro Leu Phe Val Gly SerVal Arg Asn Pro Asn Pro Ser Ala Pro Arg Glu Leu Lys Glu Gln GlnAsp Ser Pro Gly Asn Lys Asp Phe Leu Gln Ser Leu Lys Gly Phe ProArg Gly Asp Lys Leu Phe Gly Pro Asp Leu Lys Leu Val Pro Pro MetGlu Glu Asp Tyr Pro Gln Phe Gly Ser Pro Lys SEQ ID NO: 2Oryctolagus cuniculus A2AP; AA sequenceMet Val Leu Leu Arg Gly Leu Leu Val Leu Ser Leu Ser Cys Leu GlnGly Pro Cys Ala Val Leu Pro Pro Val Ser Ala Met Glu Pro Val GlyArg Gln Leu Thr Ser Gly Gln Ser Gln Glu Lys Leu Pro Pro Leu AlaLeu Leu Lys Leu Val Asn Gln Glu Leu His Gly Gln Thr Ala Leu LysLys Ser Pro Gly Asp Cys Arg Glu Thr Pro Thr Pro Glu Gln Thr ArgArg Leu Ala Gln Ala Met Met Ala Phe Thr Thr Asp Leu Phe Ser LeuVal Val Gln Ala Ser Thr Ser Pro Asn Leu Val Leu Ser Pro Leu SerVal Ala Leu Ala Leu Ser His Leu Ala Leu Gly Ala Gln Asn His ThrLeu Gln Arg Leu Gln Gln Val Leu His Ala Asp Ser Gly Pro Cys LeuPro His Leu Leu Ser His Leu Cys Arg Asn Leu Gly Pro Gly Ala PheArg Leu Ala Ala Arg Met Tyr Leu Gln Lys Gly Phe Pro Ile Lys GluAsp Phe Leu Lys Leu Ser Glu Gln Leu Phe Gly Ala Lys Pro Val SerLeu Thr Gly Arg Gln Glu Glu Asp Leu Val Asn Ile Asn Gln Trp ValLys Glu Ala Thr Glu Gly Lys Ile Glu Asp Phe Leu Ser Glu Leu ProAsp Ser Thr Val Leu Leu Leu Leu Asn Ala Ile His Phe Gln Gly PheTrp Arg Ser Lys Phe Asp Pro Ser Leu Thr Gln Arg Asp Ser Phe HisLeu Asp Glu Gln Phe Thr Val Pro Val Asp Met Met Gln Ala His LysTyr Pro Leu Arg Trp Phe Leu Leu Glu Gln Pro Glu Ile Gln Val AlaGln Phe Pro Phe Lys Asn Asn Met Ser Phe Val Val Leu Val Pro ThrAsn Phe Glu Trp Asn Val Ser Gln Val Leu Ser Asn Leu Ser Trp AspIle Leu His Gln Pro Ser Leu Arg Glu Arg Pro Thr Lys Val Gln LeuPro Lys Leu Leu Leu Lys His Gln Leu Asp Leu Val Thr Thr Leu SerGln Leu Gly Leu Gln Glu Leu Phe Leu Ala Pro Asp Leu Arg Gly IleSer Asp Glu Gly Leu Val Val Ser Ser Val Gln His Gln Ser Thr LeuGlu Leu Asn Glu Ala Gly Val Glu Ala Ala Ala Ala Thr Ser Thr AlaMet Ser Arg Met Ser Leu Ser Ser Phe Ser Val Asn Arg Pro Phe LeuPhe Phe Ile Leu Glu Asp Thr Ile Asp Leu Pro Ile Phe Val Gly IleVal Arg Asn Pro Asn Pro Ser Ala Gln Pro Glu Arg Lys Glu Gln GlnAsp Ser Pro Asp His Arg Asp Pro Ser Gln Pro Gln Lys Ser Phe ProHis Gly Asp Lys Leu Phe Ser Pro Asp Leu Lys Leu Ala Pro Pro SerGlu Glu Asp Tyr Pro Gln Leu Ser Ser Pro Lys SEQ ID NO: 3Macaca fascicularis A2AP; AA sequenceMet Ala Leu Phe Trp Gly Leu Leu Val Leu Ser Trp Ser Cys Leu GlnGly Pro Leu Ser Val Phe Ser Pro Val Ser Ala Met Glu Pro Leu GlyTrp Gln Leu Thr Ser Gly Pro Asn Gln Glu Lys Val Pro Pro Leu ThrLeu Leu Lys Leu Gly Asn Gln Glu Pro Gly Gly Gln Thr Ala Leu LysSer Leu Pro Gly Ile Cys Ser Arg Asp Pro Thr Pro Glu Gln Thr ArgArg Leu Ala Gln Ala Met Met Ala Phe Thr Ala Asp Leu Phe Ser LeuVal Ala Gln Thr Ser Thr Cys Pro Asn Leu Ile Leu Ser Pro Leu SerVal Ala Leu Ala Leu Ser His Leu Ala Leu Gly Ala Gln Asn His ThrLeu Gln Arg Leu Gln Gln Val Leu His Ala Gly Ser Gly Pro Cys LeuPro His Leu Leu Ser Arg Leu Cys Gln Asn Met Gly Pro Gly Ala PheArg Leu Ala Ala Arg Met Tyr Leu Gln Lys Gly Phe Pro Ile Lys GluAsp Phe Leu Glu Gln Ser Glu Arg Leu Phe Gly Ala Lys Pro Val SerLeu Thr Gly Lys Gln Glu Asp Asp Leu Ala Asn Ile Asn Gln Trp ValLys Glu Ala Thr Glu Gly Lys Ile Pro Glu Phe Leu Ser Glu Leu ProGlu Asp Thr Val Leu Leu Leu Leu Asn Ala Ile His Phe Gln Gly PheTrp Arg Ser Lys Phe Asp Pro Ser Leu Thr Gln Arg Asp Ser Phe HisLeu Asp Glu Gln Phe Thr Val Pro Val Glu Met Met Gln Ala Arg ThrTyr Pro Leu Arg Trp Phe Met Leu Glu Gln Pro Glu Ile Gln Val AlaHis Phe Pro Phe Lys Asn Asn Met Ser Phe Val Val Leu Val Pro ThrHis Phe Glu Trp Asn Val Ser Gln Val Leu Ala Asn Leu Ser Trp AspThr Leu Tyr Pro Pro Ser Val Trp Glu Arg Pro Thr Lys Val Arg LeuPro Lys Leu Tyr Leu Lys His Gln Met Asp Leu Met Ala Thr Leu SerArg Leu Gly Leu Gln Glu Leu Phe Gln Ala Pro Asp Leu Arg Gly IleSer Glu Gln Ser Leu Val Val Ser Gly Val Gln His Gln Ser Thr LeuGlu Leu Ser Glu Val Gly Val Glu Ala Ala Ala Ala Thr Ser Ile AlaMet Ser Arg Met Ser Leu Ser Ser Phe Ser Val Asn Arg Pro Phe LeuPhe Phe Ile Phe Glu Asp Thr Thr Gly Leu Pro Leu Phe Val Gly SerVal Arg Asn Pro Asn Pro Ser Ala Pro Arg Glu Leu Lys Glu Gln GlnAsp Ser Pro Gly Asp Lys Asp Phe Leu His Ser Leu Lys Ala Gly ProArg Gly Asp Lys Leu Phe Gly Pro Asp Leu Lys Leu Ala Pro Pro LeuGlu Glu Asp Tyr Pro Glu Leu Gly Ser Pro Lys SEQ ID NO: 6antibody sequence; Artificial AA Sequence Asp Tyr Ala Met SerSEQ ID NO: 7 antibody sequence; Artificial AA SequenceAla Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys GlySEQ ID NO: 8 antibody sequence; Artificial AA SequenceAla Ile Gly Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys GlySEQ ID NO: 9 antibody sequence; Artificial AA SequenceThr Gly Ser Ser Ser Asn Ile Gly Ala Thr Tyr Asp Val His SEQ ID NO: 10antibody sequence; Artificial AA Sequence Ser Asn Asn Gln Arg Pro SerSEQ ID NO: 11 antibody sequence; Artificial AA SequenceGlu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr SEQ ID NO: 12antibody sequence; Artificial AA SequenceGlu Asp Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr SEQ ID NO: 13antibody sequence; Artificial AA SequenceGlu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr SEQ ID NO: 14antibody sequence; Artificial AA SequenceGlu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Val Tyr SEQ ID NO: 15antibody sequence; Artificial AA SequenceGlu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Glu Tyr SEQ ID NO: 16antibody sequence; Artificial AA SequenceGlu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Thr Tyr SEQ ID NO: 17antibody sequence; Artificial AA SequenceAla Ala Trp Asp Asp Ser Leu Ser Gly Trp Val SEQ ID NO: 18antibody sequence; Artificial AA SequenceAla Ala Trp Asp Trp Ser Leu Ser Gly Trp Val SEQ ID NO: 19antibody sequence; Artificial AA SequenceTrp Ala Trp Asp Asp Ser Leu Ser Gly Trp Val SEQ ID NO: 20antibody sequence; Artificial AA SequenceAla Ala Trp Asp Val Ser Leu Ser Gly Trp Val SEQ ID NO: 21antibody sequence; Artificial AA Sequence Ser Tyr Ala Met SerSEQ ID NO: 22 antibody sequence; Artificial AA SequenceAla Ile Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys GlySEQ ID NO: 23 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 24antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AspArg Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 25antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaSer Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 26antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Asp Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 27antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 28antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Val Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 29antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Glu Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 30antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Thr Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 31antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 32antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 33antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaLys Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 34antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 35antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Ser TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 36antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 37antibody sequence; Artificial AA SequenceGln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly GlnArg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala ThrTyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys LeuLeu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg PheSer Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly LeuArg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp SerLeu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val LeuSEQ ID NO: 38 antibody sequence; Artificial AA SequenceGln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly GlnArg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala ThrTyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys LeuLeu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg PheSer Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly LeuArg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Trp SerLeu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val LeuSEQ ID NO: 39 antibody sequence; Artificial AA SequenceGln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly GlnArg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala ThrTyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys LeuLeu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg PheSer Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly LeuArg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Trp Ala Trp Asp Asp SerLeu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val LeuSEQ ID NO: 40 antibody sequence; Artificial AA SequenceGln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly GlnArg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala ThrTyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys LeuLeu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg PheSer Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly LeuArg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Val SerLeu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val LeuSEQ ID NO: 41 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlySEQ ID NO: 42 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AspArg Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu SerSEQ ID NO: 43 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaSer Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu SerSEQ ID NO: 44 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Asp Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu SerSEQ ID NO: 45 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu SerSEQ ID NO: 46 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu SerSEQ ID NO: 47 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Glu Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlySEQ ID NO: 48 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Leu Thr Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlySEQ ID NO: 49 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlySEQ ID NO: 50 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlySEQ ID NO: 51 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaLys Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlySEQ ID NO: 52 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlySEQ ID NO: 53 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Ser TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlySEQ ID NO: 54 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlySEQ ID NO: 55 antibody sequence; Artificial AA SequenceGlu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp TyrAla Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Ala Ile Gly Thr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Glu Gly Tyr Asp Ser Ser Gly Tyr Tyr His Leu Asp Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp HisLys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr GlyPro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro SerVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp ProGlu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val ValSer Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu TyrLys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys ThrIle Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr LeuPro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr CysLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerAsn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu AspSer Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys SerArg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu AlaLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu GlySEQ ID NO: 56 antibody sequence; Artificial AA SequenceGln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly GlnArg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala ThrTyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys LeuLeu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg PheSer Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly LeuArg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp SerLeu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu GlyGln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser GluGlu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp PheTyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro ValLys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn LysTyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys SerHis Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val GluLys Thr Val Ala Pro Thr Glu Cys Ser SEQ ID NO: 57antibody sequence; Artificial AA SequenceGln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly GlnArg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala ThrTyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys LeuLeu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg PheSer Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly LeuArg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Trp SerLeu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu GlyGln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser GluGlu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp PheTyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro ValLys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn LysTyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys SerHis Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val GluLys Thr Val Ala Pro Thr Glu Cys Ser SEQ ID NO: 58antibody sequence; Artificial AA SequenceGln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly GlnArg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala ThrTyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys LeuLeu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg PheSer Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly LeuArg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Trp Ala Trp Asp Asp SerLeu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu GlyGln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser GluGlu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp PheTyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro ValLys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn LysTyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys SerHis Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val GluLys Thr Val Ala Pro Thr Glu Cys Ser SEQ ID NO: 59antibody sequence; Artificial AA SequenceGln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly GlnArg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala ThrTyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys LeuLeu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg PheSer Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly LeuArg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Val SerLeu Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu GlyGln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser GluGlu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp PheTyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro ValLys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn LysTyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys SerHis Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val GluLys Thr Val Ala Pro Thr Glu Cys Ser SEQ ID NO: 60 DNA; Homo sapiensatggcgctgc tctgggggct cctggtgctc agctggtcct gcctgcaagg cccctgctcc 60gtgttctccc ctgtgagcgc catggagccc ttgggccggc agctaactag cgggccgaac 120caggagcagg tgtccccact taccctcctc aagttgggca accaggagcc tggtggccag 180actgccctga agagtccccc aggagtctgc agcagagacc ccaccccaga gcagacccac 240aggctggccc gggccatgat ggccttcact gccgacctgt tctccctggt ggctcaaacg 300tccacctgcc ccaacctcat cctgtcaccc ctgagtgtgg ccctggcgct gtctcacctg 360gcactaggtg ctcagaacca cacgttgcag aggctgcaac aggtgctgca cgcaggctca 420gggccctgcc tcccccatct gctgagccgc ctctgccagg acctgggccc cggcgcgttc 480cgactggctg ccaggatgta cctgcagaaa ggatttccca tcaaagaaga tttcctggaa 540caatccgaac agctatttgg ggcaaagccc gtgagcctga cgggaaagca ggaagatgac 600ctggcaaaca tcaaccaatg ggtgaaggag gccacggagg ggaagattca ggaattcctc 660tctgggctgc cggaagacac cgtgttgctt ctcctcaacg ccatccactt ccagggtttc 720tggaggaaca agtttgaccc gagccttacc cagagagact ccttccacct ggacgagcag 780ttcacggtgc ccgtggaaat gatgcaggcc cgcacgtacc cgctgcgctg gttcttgctg 840gagcagcctg agatccaggt ggctcatttc ccctttaaga acaacatgag ctttgtggtc 900cttgtaccca cccactttga atggaacgtg tcccaggtac tggccaacct gagttgggac 960accctgcacc cacctctggt gtgggagagg cccaccaagg tccggctgcc taagctgtat 1020ctgaaacacc aaatggacct ggtggccacc ctcagccagc tgggcctgca ggagttgttc 1080caggccccag acctgcgtgg gatctccgag cagagcctgg tggtgtccgg cgtgcagcat 1140cagtccaccc tggagctcag cgaggtcggc gtggaggcgg cggcggccac cagcattgcc 1200atgtcccgca tgtccctgtc ctccttcagc gtgaaccgcc ccttcctctt cttcatcttc 1260gaggacacca caggccttcc cctcttcgtg ggcagcgtga ggaaccccaa ccccagtgca 1320ccgcgggagc tcaaggaaca gcaggattcc ccgggcaaca aggacttcct ccagagcctg 1380aaaggcttcc cccgcggaga caagcttttc ggccctgact taaaacttgt gccccccatg 1440gaggaggatt acccccagtt tggcagcccc aagtga 1476 SEQ ID NO: 61DNA; Oryctolagus cuniculusatggtgctgc tccgggggct cctggtgctc agcttgtcct gcctgcaagg cccctgcgcg 60gtgctccctc ccgtgagcgc catggagccc gtgggccggc agctaactag tggtcagagc 120caagaaaagc tgcctccgct cgccctccto aagttggtca accaggagct gcacggtcag 180actgccctga agaagtcccc aggagactgc agggaaaccc cgaccccgga gcagacgcgc 240aggctggcgc aggccatgat ggccttcacc actgacctgt tttccctggt ggtgcaagca 300tccaccagcc ccaacctggt cttgtcgccc ctgagtgtgg ccctggctct gtctcacctg 360gcattaggtg ctcagaacca cacgctacag aggttgcagc aggtgctgca tgcggactca 420gggccctgcc tcccccacct gctgagccac ctctgccgga acctgggccc aggggcgttc 480cgattggctg ccagaatgta cctgcagaaa ggctttccca tcaaagagga cttcctgaag 540ctgtcagagc agctgtttgg tgcaaagcct gtgagcctga caggaaggca agaggaggac 600ctggtgaaca tcaatcaatg ggtgaaggag gccacagagg ggaagattga ggatttcctc 660tcggaattgc cagacagcac cgtgctgctc ctcctcaatg ccatccactt ccagggtttc 720tggaggagca aatttgaccc gagcctcacc cagagagact ccttccacct ggacgagcag 780ttcacggtgc cagtggacat gatgcaagcc cacaagtacc ctctgcgctg gttcttgctg 840gagcagcctg agatccaggt ggcccaattc ccctttaaga acaacatgag ctttgtggtc 900ctcgtgccca cgaactttga gtggaacgtg tcccaggtgc tgagcaacct gagctgggac 960atcctgcacc agccctcact gcgggagagg cccaccaaag tccagctgcc caagctgctc 1020ctgaaacacc agctggacct ggtgaccacc ctcagccagc tgggcctgca ggagctgttc 1080ctggccccag acctgcgtgg gatctccgac gagggcctgg tggtgtccag tgtacaacat 1140cagtccaccc tggagctcaa cgaggctggt gtggaggcgg ccgcggccac cagcacggcc 1200atgtcgcgca tgtccctttc ctccttcagc gtgaaccgcc ccttcctctt cttcatcctg 1260gaggacacca tagacctgcc catctttgtg ggcatagtgc ggaaccccaa tcctagcgcg 1320cagccagagc gcaaggagca gcaggattcc cctgaccaca gggacccctc gcagccccag 1380aaatccttcc cccacgggga caagctcttc agccccgact tgaaactggc gcccccgtcg 1440gaagaggatt acccccagct cagcagcccc aagtga 1476 SEQ ID NO: 62DNA; Macaca fascicularisatggcgctgt tctgggggct cctggtgctc agctggtcct gcctgcaagg tcccctctcc 60gtgttctccc ctgtgagcgc catggagccc ttgggctggc agctaactag tgggccaaac 120caagagaagg tgcccccact tactctcctc aagttgggca accaggagcc tggcggccag 180actgccctga agagtctccc aggaatctgc agcagagacc ccacccccga gcagacccgc 240aggctggccc aggccatgat ggccttcact gccgacctgt tctccctggt ggctcaaacg 360tccacctgcc ccaacctcat cctgtcacct ctgagtgtgg ccctggcgct gtctcacctg 300gcactaggtg ctcagaacca cacgctgcag aggctgcaac aggtgctgca cgcaggctca 420gggccctgcc taccccatct gctgagccgc ctctgccaga acatgggccc cggggccttc 480cgactggctg ccaggatgta cctgcagaaa ggatttccca tcaaagaaga tttcctggaa 540cagtctgaac ggctatttgg ggcaaagccc gtgagcctga cgggaaagca ggaagatgac 600ctggcaaaca tcaaccaatg ggtgaaggag gccacggagg ggaagattcc ggagttcctc 660tctgagctac cggaagacac cgtgttgctt ctcctcaacg ccatccactt ccagggtttc 720tggaggagca agtttgaccc gagcctcacc cagagagact ccttccacct ggacgagcag 780ttcacggtgc ccgtggaaat gatgcaagcc cgcacgtatc ctctgcgctg gttcatgctg 840gagcagcccg agatccaggt ggctcatttt ccctttaaga acaacatgag ctttgtggtc 900cttgtaccca cccactttga atggaacgtg tcccaggtac tggccaacct gagttgggac 960accctgtacc caccttccgt gtgggagagg cccaccaagg tccggctgcc taagctgtat 1020ctgaaacacc aaatggacct gatggccacc ctcagccggc tgggcctgca ggagctgttc 1080caggccccag acctgcgcgg gatctctgag cagagcctgg tggtgtccgg cgtgcagcat 1140cagtccaccc tggagctcag cgaggtcggc gtggaggcgg cggcggccac cagcatcgcc 1200atgtcccgca tgtccctgtc ctccttcagc gtgaaccgcc ccttcctctt cttcatcttt 1260gaggacacca caggccttcc cctctttgtg ggcagcgtga ggaaccccaa ccccagcgcg 1320ccacgggagc tcaaggagca gcaggattcc ccgggagaca aggacttcct ccacagcctg 1380aaagccggcc cccgcggaga caagctcttc ggccctgact tgaaacttgc gccccccttg 1440gaggaggatt accctgagct tggcagccct aagtga 1476 SEQ ID NO: 63DNA; Artificial Sequence, antibody sequence gattacgcca tgagc 15SEQ ID NO: 64 DNA; Artificial Sequence, antibody sequencegccatcggaa caggcggcgg aacatattac gccgacagcg tgaagggc 48 SEQ ID NO: 65DNA; Artificial Sequence, antibody sequencegccatcggca caggcggcag cacatattac gccgactctg tgaagggc 48 SEQ ID NO: 66DNA; Artificial Sequence, antibody sequenceaccggcagca gctccaatat cggcgccacc tatgacgtgc ac 42 SEQ ID NO: 67DNA; Artificial Sequence, antibody sequence agcaacaacc agcggcctag c 21SEQ ID NO: 68 DNA; Artificial Sequence, antibody sequencegagtactacg acagcagcgg ctactaccac ctggactat 39 SEQ ID NO: 69DNA; Artificial Sequence, antibody sequencegaggattacg acagcagcgg ctactaccac ctggactat 39 SEQ ID NO: 70DNA; Artificial Sequence, antibody sequencegaggggtacg acagcagcgg ctactaccac ctggactat 39 SEQ ID NO: 71DNA; Artificial Sequence, antibody sequencegagtactacg acagcagcgg ctactaccac ctggtttat 39 SEQ ID NO: 72DNA; Artificial Sequence, antibody sequencegagtactacg acagcagcgg ctactaccac ctggagtat 39 SEQ ID NO: 73DNA; Artificial Sequence, antibody sequencegagtactacg acagcagcgg ctactaccac ctgacgtat 39 SEQ ID NO: 74DNA; Artificial Sequence, antibody sequencegccgcctggg atgattctct gagcggctgg gtt 33 SEQ ID NO: 75DNA; Artificial Sequence, antibody sequencegccgcctggg attggtctct gagcggctgg gtt 33 SEQ ID NO: 76DNA; Artificial Sequence, antibody sequencetgggcctggg atgattctct gagcggctgg gtt 33 SEQ ID NO: 77DNA; Artificial Sequence, antibody sequencegccgcctggg atgtttctct gagcggctgg gtt 33 SEQ ID NO: 78DNA; Artificial Sequence, antibody sequence agctacgcca tgagc 15SEQ ID NO: 79 DNA; Artificial Sequence, antibody sequencegccatcggca gcggaggcag cacatattac gccgactctg tgaagggc 48 SEQ ID NO: 80DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agagtactac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tca 363 SEQ ID NO: 81 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgatag agagtactac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tca 363 SEQ ID NO: 82 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag tgagtactac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tca 363 SEQ ID NO: 83 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agaggattac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tca 363 SEQ ID NO: 84 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agaggggtac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tca 363 SEQ ID NO: 85 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agagtactac 300gacagcagcg gctactacca cctggtttat tggggccagg gcaccctggt cacagtttct 360tca 363 SEQ ID NO: 86 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agagtactac 300gacagcagcg gctactacca cctggagtat tggggccagg gcaccctggt cacagtttct 360tca 363 SEQ ID NO: 87 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agagtactac 300gacagcagcg gctactacca cctgacgtat tggggccagg gcaccctggt cacagtttct 360tca 363 SEQ ID NO: 88 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt cacctttagc gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tca 363 SEQ ID NO: 89 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggcacag gcggcagcac atattacgcc 180gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tca 363 SEQ ID NO: 90 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact actgtgccaa agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tca 363 SEQ ID NO: 91 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt cacctttagc gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggcacag gcggcagcac atattacgcc 180gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tca 363 SEQ ID NO: 92 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt cacctttgat agctacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggcacag gcggcagcac atattacgcc 180gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tca 363 SEQ ID NO: 93 DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggcagcg gaggcagcac atattacgcc 180gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tca 363 SEQ ID NO: 94 DNA; Artificial Sequence, antibody sequencecagtctgttc tgacacagcc tcctagcgcc tctggcacac ctggacagag agtgaccatc 60agctgtaccg gcagcagctc caatatcggc gccacctatg acgtgcactg gtatcagcag 120ctgcctggca cagcccctaa actgctgatc tacagcaaca accagcggcc tagcggcgtg 180cccgatagat tttctggcag caagagcggc acaagcgcca gcctggctat ctctggactg 240agatctgagg acgaggccga ctactattgc gccgcctggg atgattctct gagcggctgg 300gttttcggcg gaggcacaaa actgacagtg cta 333 SEQ ID NO: 95DNA; Artificial Sequence, antibody sequencecagtctgttc tgacacagcc tcctagcgcc tctggcacac ctggacagag agtgaccatc 60agctgtaccg gcagcagctc caatatcggc gccacctatg acgtgcactg gtatcagcag 120ctgcctggca cagcccctaa actgctgatc tacagcaaca accagcggcc tagcggcgtg 180cccgatagat tttctggcag caagagcggc acaagcgcca gcctggctat ctctggactg 240agatctgagg acgaggccga ctactattgc gccgcctggg attggtctct gagcggctgg 300gttttcggcg gaggcacaaa actgacagtg cta 333 SEQ ID NO: 96DNA; Artificial Sequence, antibody sequencecagtctgttc tgacacagcc tcctagcgcc tctggcacac ctggacagag agtgaccatc 60agctgtaccg gcagcagctc caatatcggc gccacctatg acgtgcactg gtatcagcag 120ctgcctggca cagcccctaa actgctgatc tacagcaaca accagcggcc tagcggcgtg 180cccgatagat tttctggcag caagagcggc acaagcgcca gcctggctat ctctggactg 240agatctgagg acgaggccga ctactattgc tgggcctggg atgattctct gagcggctgg 300gttttcggcg gaggcacaaa actgacagtg cta 333 SEQ ID NO: 97DNA; Artificial Sequence, antibody sequencecagtctgttc tgacacagcc tcctagcgcc tctggcacac ctggacagag agtgaccatc 60agctgtaccg gcagcagctc caatatcggc gccacctatg acgtgcactg gtatcagcag 120ctgcctggca cagcccctaa actgctgatc tacagcaaca accagcggcc tagcggcgtg 180cccgatagat tttctggcag caagagcggc acaagcgcca gcctggctat ctctggactg 240agatctgagg acgaggccga ctactattgc gccgcctggg atgtttctct gagcggctgg 300gttttcggcg gaggcacaaa actgacagtg cta 333 SEQ ID NO: 98DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agagtactac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgoctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 99DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgatag agagtactac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 100DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag tgagtactac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgoctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 101DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agaggattac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 102DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agaggggtac 300gacagcagcg gctactacca cctggactat tggggccagg gcaccctggt cacagtttct 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 103DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agagtactac 300gacagcagcg gctactacca cctggtttat tggggccagg gcaccctggt cacagtttct 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 104DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agagtactac 300gacagcagcg gctactacca cctggagtat tggggccagg gcaccctggt cacagtttct 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 105DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgcgccag agagtactac 300gacagcagcg gctactacca cctggagtat tggggccagg gcaccctggt cacagtttct 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccato 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 106DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt cacctttagc gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 107DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggcacag gcggcagcac atattacgcc 180gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 108DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggaacag gcggcggaac atattacgcc 180gacagcgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact actgtgccaa agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 109DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt cacctttagc gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggcacag gcggcagcac atattacgcc 180gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 110DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt cacctttgat agctacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggcacag gcggcagcac atattacgcc 180gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 111DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggcagcg gaggcagcac atattacgcc 180gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tcagccagca ccaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct 420ggcggaacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagctctct gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct gcgacaagac ccacacctgt cccccttgtc ctgcccccga actgctggga 720ggcccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat cagccggacc 780cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 840tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900aacagcacct accgggtggt gtccgtgctg acagtgctgc accaggactg gctgaacggc 960aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga gaaaaccatc 1020agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc aagcagggac 1080gagctgacca agaaccaggt gtccctgacc tgtctcgtga aaggcttcta cccctccgat 1140atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac caccccccct 1200gtgctggaca gcgacggctc attcttcctg tacagcaagc tgaccgtgga caagtcccgg 1260tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320acccagaagt ccctgagcct gagccctggc 1350 SEQ ID NO: 112DNA; Artificial Sequence, antibody sequencegaagttcagc tgctggaatc tggcggcgga ctggttcaac ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcgat gattacgcca tgagctgggt ccgacaggcc 120cctggaaaag gccttgaatg ggtgtccgcc atcggcacag gcggcagcac atattacgcc 180gactctgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240cagatgaaca gcctgagagc cgaggacacc gccgtgtact attgtgccag agagggctac 300gacagcagcg gctactacca cctggattat tggggccagg gcaccctggt tacagtgtca 360tcagccagca ccaagggccc cagcgtgttc cctctggccc cttgtagcag aagcaccagc 420gagtctacag ccgccctggg ctgcctcgtg aaggactact ttcccgagcc cgtgaccgtg 480tcctggaact ctggcgctct gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtcgtg acagtgccca gcagcagcct gggcaccaag 600acctacacct gtaacgtgga ccacaagccc agcaacacca aggtggacaa gcgggtggaa 660tctaagtacg gccctccctg ccctccttgc ccagcccctg aatttctggg cggaccctcc 720gtgttcctgt tccccccaaa gcccaaggac accctgatga tcagccggac ccccgaagtg 780acctgcgtgg tggtggatgt gtcccaggaa gatcccgagg tgcagttcaa ttggtacgtg 840gacggcgtgg aagtgcacaa cgccaagacc aagcccagag aggaacagtt caacagcacc 900taccgggtgg tgtccgtgct gacagtgctg caccaggact ggctgaacgg caaagagtac 960aagtgcaagg tgtccaacaa gggcctgccc agctccatcg agaaaaccat cagcaaggcc 1020aagggccagc cccgcgaacc ccaggtgtac acactgcctc caagccagga agagatgacc 1080aagaaccagg tgtccctgac ctgtctcgtg aaaggcttct acccctccga tatcgccgtg 1140gaatgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc tgtgctggac 1200agcgacggct cattcttcct gtacagcaga ctgaccgtgg acaagagccg gtggcaggaa 1260ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320tccctgtctc tgagcctggg c 1341 SEQ ID NO: 113DNA; Artificial Sequence, antibody sequenceagtctgttc tgacacagcc tcctagcgcc tctggcacac ctggacagag agtgaccatc 60agctgtaccg gcagcagctc caatatcggc gccacctatg acgtgcactg gtatcagcag 120ctgcctggca cagcccctaa actgctgatc tacagcaaca accagcggcc tagcggcgtg 180cccgatagat tttctggcag caagagcggc acaagcgcca gcctggctat ctctggactg 240agatctgagg acgaggccga ctactattgc gccgcctggg atgattctct gagcggctgg 300gttttcggcg gaggcacaaa actgacagtg ctaggccagc ctaaagccgc ccctagcgtg 360accctgttcc ctccaagcag cgaggaactg caggccaaca aggccaccct cgtgtgcctg 420atcagcgact tctatcctgg cgccgtgacc gtggcctgga aggccgatag ctctcctgtg 480aaggccggcg tggaaaccac cacccctagc aagcagagca acaacaaata cgccgccagc 540agctacctga gcctgacccc cgagcagtgg aagtcccaca gatcctacag ctgccaagtg 600acccacgagg gcagcaccgt ggaaaagaca gtggccccta ccgagtgcag c 651SEQ ID NO: 114 DNA; Artificial Sequence, antibody sequencecagtctgttc tgacacagcc tcctagcgcc tctggcacac ctggacagag agtgaccatc 60agctgtaccg gcagcagctc caatatcggc gccacctatg acgtgcactg gtatcagcag 120ctgcctggca cagcccctaa actgctgatc tacagcaaca accagcggcc tagcggcgtg 180cccgatagat tttctggcag caagagcggc acaagcgcca gcctggctat ctctggactg 240agatctgagg acgaggccga ctactattgc gccgcctggg attggtctct gagcggctgg 300gttttcggcg gaggcacaaa actgacagtg ctaggccagc ctaaagccgc ccctagcgtg 360accctgttcc ctccaagcag cgaggaactg caggccaaca aggccaccct cgtgtgcctg 420atcagcgact tctatcctgg cgccgtgacc gtggcctgga aggccgatag ctctcctgtg 480aaggccggcg tggaaaccac cacccctagc aagcagagca acaacaaata cgccgccagc 540agctacctga gcctgacccc cgagcagtgg aagtcccaca gatcctacag ctgccaagtg 600acccacgagg gcagcaccgt ggaaaagaca gtggccccta ccgagtgcag c 651SEQ ID NO: 115 DNA; Artificial Sequence, antibody sequencecagtctgttc tgacacagcc tcctagcgcc tctggcacac ctggacagag agtgaccatc 60agctgtaccg gcagcagctc caatatcggc gccacctatg acgtgcactg gtatcagcag 120ctgcctggca cagcccctaa actgctgatc tacagcaaca accagcggcc tagcggcgtg 180cccgatagat tttctggcag caagagcggc acaagcgcca gcctggctat ctctggactg 240agatctgagg acgaggccga ctactattgc tgggcctggg atgattctct gagcggctgg 300gttttcggcg gaggcacaaa actgacagtg ctaggccagc ctaaagccgc ccctagcgtg 360accctgttcc ctccaagcag cgaggaactg caggccaaca aggccaccct cgtgtgcctg 420atcagcgact tctatcctgg cgccgtgacc gtggcctgga aggccgatag ctctcctgtg 480aaggccggcg tggaaaccac cacccctagc aagcagagca acaacaaata cgccgccagc 540agctacctga gcctgacccc cgagcagtgg aagtcccaca gatcctacag ctgccaagtg 600acccacgagg gcagcaccgt ggaaaagaca gtggccccta ccgagtgcag c 651SEQ ID NO: 116 DNA; Artificial Sequence, antibody sequencecagtctgttc tgacacagcc tcctagcgcc tctggcacac ctggacagag agtgaccatc 60agctgtaccg gcagcagctc caatatcggc gccacctatg acgtgcactg gtatcagcag 120ctgcctggca cagcccctaa actgctgatc tacagcaaca accagcggcc tagcggcgtg 180cccgatagat tttctggcag caagagcggc acaagcgcca gcctggctat ctctggactg 240agatctgagg acgaggccga ctactattgc gccgcctggg atgtttctct gagcggctgg 300gttttcggcg gaggcacaaa actgacagtg ctaggccagc ctaaagccgc ccctagcgtg 360accctgttcc ctccaagcag cgaggaactg caggccaaca aggccaccct cgtgtgcctg 420atcagcgact tctatcctgg cgccgtgacc gtggcctgga aggccgatag ctctcctgtg 480aaggccggcg tggaaaccac cacccctagc aagcagagca acaacaaata cgccgccagc 540agctacctga gcctgacccc cgagcagtgg aagtcccaca gatcctacag ctgccaagtg 600acccacgagg gcagcaccgt ggaaaagaca gtggccccta ccgagtgcag c 651SEQ ID NO: 117 DNA; Artificial Sequence, antibody sequencegagggctacg acagcagcgg ctactaccac ctggattat 39 SEQ ID NO: 118human plasmin; AA sequenceGlu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe SerVal Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala AlaLys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr HisSer Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser SerIle Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val TyrLeu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr MetSer Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr SerPro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly LeuGlu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro TrpCys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile LeuGlu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp GlyLys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp SerGln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn LysAsn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg ProTrp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp IlePro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln CysLeu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr ValSer Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr HisAsn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn TyrCys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr AsnSer Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser SerPro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu ThrPro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg GlyThr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser SerMet Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn AlaGly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly ProTrp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn LeuLys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro ValVal Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met PheGly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr GlyThr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser IlePhe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr CysArg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr AsnPro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala ProSer Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly ArgSEQ ID NO: 115 human plasmin; AA sequenceVal Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln ValSer Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu IleSer Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser ProArg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val AsnLeu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu GluPro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala ValIle Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr ValVal Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr GlnGly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val IleGlu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val GlnSer Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser CysGln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys TyrIle Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro AsnLys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile GluGly Val Met Arg Asn Asn

EXAMPLES Example 1: Generation of Antibody TPP-12387 from BioInventAntibody Library

A fully human antibody phage display library (BioInvent n-CoDeR Fablambda library) was used to isolate human monoclonal antibodies byselection against soluble biotinylated antigens, which are humanalpha2-Antiplasmin from human and from rabbit origin.

The human alpha2-Antiplasmin was used from a commercial source(antibodies online; catalogue number ABIN2544306) whereas the rabbitantigen was produced in house by recombinant expression andpurification. For this, the cDNA from rabbit alpha2-Antiplasmin wascloned into standard expression vector and HEK293 cells were transientlytransfected with this construct using 293fectin transfection reagent(Invitrogen, catalogue number 12347-019) following manufacturesinstructions. Expressed rabbit alpha2-Antiplasmin were purified from thecell culture supernatant via Ni-IMAC and size exclusion chromatography.

Antigens were biotinylated using a Sulfo-NHS-LC-Biotin kit (ThermoScientific, catalogue number A39257). Free biotin was removed from thereactions by dialysis against the appropriate buffer.

For the panning procedure the following protocol was applied:Streptavidin-coupled Dynabeads M-280 (Invitrogen, catalogue number11205D) were coated for one hour at room temperature (RT) with thebiotinylated antigen (1 tube) and the biotinylated off-target (3 tubes),respectively. Dynabeads were washed and subsequently blocked for 1 h atRT with end-over-end rotation. For depletion of off-target binders theblocked phage library was added to the blocked off-target loadedDynabeads and incubated for 10 min at room temperature with end-over-endrotation. This depletion step was repeated 2 times. The depleted phagelibrary was added to the blocked target loaded Dynabeads and incubatedfor 60 min at RT with end-over-end rotation. After stringent washing (3×in blocking buffer and 9× in PBS (150 mM NaCl; 8 mM Na2HPO4; 1.5 mMKH2PO4; adjusted to pH=7.4-7.6) with 0.05% Tween-20) Dynabeads withFab-phages binding specifically to the coated target were directly usedto infect Escherichia coli strain HB101. Subsequently the phages areamplified in Escherichia coli strain HB101 using M13K07 Helper Phage(Invitrogen, catalogue number 18311019). In the following selectionrounds the target concentration was decreased to augment the selectionpressure for high affinity binders.

During panning of this library, four different selection strategies werecarried out:

Strategy I was designed in such a way to identify antibodies exhibitingbinding activity towards the full length human alpha2-Antiplasmin andrabbit alpha2-Antiplasmin, respectively, both lacking the N-terminus. Adepletion step was included using a biotinylated irrelevant protein.

Strategy II aimed for antibodies recognizing Plasmin binding site ofalpha2-Antiplasmin. Like in Strategy I and in order to increase theprobability of success, a depletion step was included using analpha2-Antiplasmin variant missing the Plasmin-binding site as antigen.

In two further strategies (strategy III and IV) a biotinylated linearand a biotinylated cyclic peptide, respectively, representing theso-called Reactive Center Loop (RCL) of alpha2-Antiplasmin, were used.In both cases, an irrelevant biotinylated protein was used for thedepletion step.

A detailed overview of the panning strategies is given in FIG. 1 .

Example 2: Recombinant DNA Constructs and Expression, Purification andQuantification of Fabs and Full-Length Antibodies

Production in HEK293-6E Cells

Fabs as well as full length antibodies were produced by mammalian cellculture using transiently transfected HEK293-6E cells. Heavy and lightchains were cloned into a suitable expression vector system. Cells wereincubated for 3-4 days. Supernatants were collected and Fabs andantibodies were purified as described.

Purification and Quantification of Fabs and Antibodies

Antibodies were purified by Protein A chromatography (ThermoFischer,catalogue number A26455) according to manufacturer's instructions.

The antibodies, antigen binding portions, or derivatives thereof wererecovered from the culture medium by using standard protein purificationmethods.

Fabs were purified from sterile filtered mammalian cell supernatantsusing a 3-step research downstream process. As capture step a “CaptureSelect IgG-CH1” affinity column (ThermoFisher, catalogue number494320005) equilibrated in PBS pH 7.4 was used. After washing in washbuffer (PBS pH 7.4) for 10 column volumes, elution of the Fab wasachieved using glycine 0.1M pH 3.0 (6 CV). Upon neutralization with TrisBase a size exclusion chromatography (GE Healthcare, Superdex 200,catalogue number GE29321905) was used for buffer exchange into DPBS pH7.4 and aggregate removal. Analytical size exclusion chromatographydemonstrated that no dimer was present in the resulting batch.

For quantification of full-length antibodies, the anti-human IgG Fcspecific antibody (Sigma, catalogue number 12136) was coated at aconcentration of 5 μg/ml over night at 4° C. to 384-well microtiterplates (Nunc). Solutions containing the IgGs of interest were added atdifferent concentrations an incubated for 1 hour at room temperature.For detection, the detection antibody A0170 (Sigma) and as substrateAmplex Red were added. Fluorescence was monitored at 535/590 nm using aSpectraFluorplus Reader (Tecan).

For quantification of antibody variants like Fabs, the Human Kappa ELISAKit (Abcam, catalogue number ab157709) was used according to themanufacturer's instructions.

Example 3: Enzyme-Linked Immunosorbent Assay (ELISA) for TestingAnti-Alpha2-Antiplasmin Binding Activity

A standard ELISA format was used for analyzing the binding affinity ofFabs of this invention to human and rabbit alpha-2-Antiplasmin,respectively. Antigens were coated to black 384 well Maxisorp microtiterplates (Nunc; catalogue number 460518), diluted to a concentration of 1μg/ml in 1× Coating Buffer (Candor Bioscience; catalogue number 121125).Plates were incubated overnight at 4° C. After overnight incubation,plates were washed 2× with 50 μl/well using PBS+0.05% Tween 20.Following this, 50 μl/well of blocking buffer (Smart Block; CandorBioscience; catalogue number 113500) was added and the plates wereincubated for 1 hour at room temperature. Afterwards, plates were washedfor 3× using well of a PBS+0.05% Tween 20 buffer. Fabs of this inventionwere added at different concentrations in a final volume of 30 μl/well.Plates were incubated for 1 hour at room temperature. Following thisincubation step, plates were washed for 3× using 50 μl/well of aPBS+0.05% Tween 20 buffer. For the detection of bound Fabs andfull-length antibodies, the anti-Human Lambda Light Chains (Bound andFree)—Peroxidase antibody (Sigma; catalogue number A5175) was diluted bythe factor of 1:10.000 in 10% Blocking Buffer. 30 μl/well of thisdiluted detection antibody was added and plates are incubated for 1 hourat room temperature. Following this incubation step, plates were washedfor 3× using 50 μl/well of a PBS+0.05% Tween 20 buffer. As substrate, amixture of 30 μl/well of 1:1000 diluted Amplex red (Invitrogen;catalogue number 12222; stock solution 10 mM in DMSO) and 1:10.000 ofHydrogen peroxide (Merck; catalogue number 107209; 30% stock solution)was added and the plates incubated for minutes in the dark.

For measurement, the Infinite f500 reader (Tecan) was used. Measurementmode: Fluorescence; Top reading; Ex 535 nm; Em 590 nm.

From an overall number of 2×10¹⁰ Fab variants screened, 2944 variantswere selected as potential candidates to be tested for binding human andrabbit alpha2-Antiplasmin, respectively.

As described in Example 2, HEK293 cells were transiently transfected.Resulting supernatants were used directly without further purificationor dilution for testing their ability to bind the human and rabbitantigen. From these 2944 Fabs, 88 candidates exhibiting distinctsequences showed the requested cross-species binding activity towardsthe two antigens.

For conformation, 88 variants of this invention were re-tested for theirbinding capacity. For this, supernatants of transfected cells werediluted in Phosphate-buffered Saline (PBS) by the factor of 1:1.5,1:4.5, 1:13.5, 1:40.5, 1:121.5, 1:364.5, 1:1093.5. These diluted sampleswere tested for binding human and rabbit alpha-2-Antiplasmin.

One dose response curve is shown in FIG. 4 as an example from two tothree independent experiments performed in quadruplicate. For the threeindependent experiments, the EC50 value for the binding activity ofTPP-12387 towards human alpha2-Antiplasmin were as follows: 1.2E-07 M(as shown in FIG. 4 ), 1.0E-07 M, and 1.3E-07 M, respectively. Thebinding activity towards rabbit alpha2-Antiplasmin were 6.0E-09 M (asshown in FIG. 4 ), 6.07E-09 M and 6.2E-09 M, respectively.

Example 4: Biochemical Assay for Testing Selected Candidates forAlpha2-Antiplasmin Function Blocking Activity

For testing the anti-alpha2-Antiplasmin molecules for functionalblocking activity, Fabs or full-length antibodies were pre-incubatedwith 1 nM of human alpha2-Antiplasmin (antibodies online; cataloguenumber ABIN2544306) or in house produced rabbit alpha2-Antiplasmin or inhouse produced cynomolgus alpha2-Antiplasmin in a buffer consisting of50 mM TRIS-HCl (GIBCO; catalogue number 15567-027 (50 mM)), 100 mM NaCl(Sigma; catalogue number S7653), 5 mM CaCl2 (Sigma; catalogue number21115-100ML), 0.1% Albumin 0.1% (Sigma; BSA, catalogue numberA4503-100g), pH 7.4 for minutes at 37° C.

Afterwards, human Plasmin (Haematologic Technologies INC.; cataloguenumber HCPM0140) at a final concentration of 400 pM and the fluorogenicsubstrate 1-1275 (Bachem; MeOSuc-Ala-Phe-Lys-AMC trifluoroacetate salt;catalogue number 1-1275 (stock: 10 mM in DMSO)) at a final concentrationof 50 μm were added and the reaction was incubated for 1 hour at 37° C.This reaction was carried out in 384 well microtiter plates (Nunc;catalogue number 262260). The fluorogenic signals were measured at thefollowing conditions: modus fluorescence top reading, Ex 360 nM, Em 465nm, Ex bandwidth 5 nm, Em bandwidth 5 nm.

For determination of dose dependencies of Fabs and/or full-lengthantibodies function blocking activity, the concentrations of thesemolecules were measured as described above. Different concentrations ofFabs and/or full-length antibodies starting at a defined concentration,followed by 1:3 or 1:4 dilution steps, were pre-incubated with 1 nM ofhuman, cynomolgus, or rabbit alpha2-Antiplasmin, respectively.

The 88 Fab candidates were tested for function blocking activity insingle-point measurement on human and rabbit alpha2-Antiplasmin, meaningthat from the supernatants resulting from transiently transfected HEK293cells the maximal possible volume was added to the activity assay.Hereby, 17 Fabs were identified showing at least a reduction ofalpha2-Antiplasmin activity of ca. 30%.

In a next step, these 17 Fabs were reformatted into the full length IgG1antibody format. 12 of these showed reasonable expression rates.

According to the method described in Example 3, these 12 antibodies weretested for ability to bind human and rabbit alpha2-Antiplasmin in adose-dependent manner. Data generated were analyzed using theGraphPadPrism software. The binding activities of the antibodies werecalculated as EC50 values. Two to three independent experiments wereperformed in quadruplicate.

In a next step, these 12 full length antibodies were re-testeddose-dependently for blocking the activity towards human and rabbitalpha2-Antiplasmin.

Based on its binding activity and function blocking activity towardshuman and rabbit alpha2-Antiplasmin antibody TPP-12387 was chosen forfurther testing and optimization. FIG. 2 shows the binding activity ofthe Fab fragment corresponding to TPP-12387 for human and rabbitalpha2-Antiplasmin as determined in Example 3. FIG. 3 shows the functionblocking activity of the Fab fragment corresponding to TPP-12387 towardshuman and rabbit alpha2-Antiplasmin as determined in Example 4. FIG. 4shows the binding activity of the IgG1 antibody TPP-12387 for human andrabbit alpha2-Antiplasmin as determined in a binding assay described inExample 3. FIG. 5 shows the function blocking activity of the IgG1antibody TPP-12387 towards human and rabbit alpha2-Antiplasmin asdetermined in a function blocking assay described in Example 4. One doseresponse curve for the neutralizing activity of TPP-12387 on human andrabbit alpha2-Antiplasmin is shown in FIG. 5 as an example of two tothree independent experiments performed in quadruplicate. For the threeindependent experiments, the IC50 value for the function blockingactivity of TPP-12387 towards human alpha2-Antiplasmin were as follows:1.7E-07 M (as shown in FIG. 5 ), 1.8E-07 M, and 1.8E-07 M, respectively.The binding activity towards rabbit alpha2-Antiplasmin were 1.4E-08 M(as shown in FIG. 4 ), 1.3E-08 M and 1.5E-08 M.

In a next step, TPP-12387 was tested for its binding activity as well asfor its function blocking activity towards cynomolgusalpha2-Antiplasmin. Cynomolgus alpha2-Antiplasmin was made in the sameway as the rabbit alph2-Antiplasmin. Values of the binding activity aswell as its function blocking activity of TPP-12387 towards cynomolgusalpha2-Antiplasmin are shown in FIG. 6 . One dose response curve for thebinding and function blocking activity of antibody TPP-12387 onCynomolgus alpha2-Antiplasmin is shown in FIG. 6 as example of two tothree independent experiments performed in quadruplicate. Hereby, theEC50 values for the binding activity of TPP-12387 towards Cynomolgusalpha2-Antiplasmin was 9.0E-08 M (as shown in FIGS. 6.1 ) and 9.0E-08 Min a second, independent experiment, and the IC50 for the functionblocking activity of Cynomolgus alpha2-Antiplasmin was 1.6E-07 M (asshown in FIGS. 6.2 ) and 1.6E-07 M in a second experiment.

Example 5: Affinity Optimization of Lead Antibody TPP-12387

Antibody TPP-12387 was subjected to lead optimization procedures aimingto optimize its affinity and to increase its functional efficiency.

Affinity maturation was done by a first single mutation gathering roundfollowed by recombination of the most affinity- and potency-increasingamino acid exchanges followed by germlining and sequence optimizationcampaign. For mutation gathering NNK (N=A or G or C or T, K=G or T)randomizations at the following individual amino acid positions weregenerated by site directed mutagenesis using synthetic oligonucleotidesincluding NNK codon-diversification of residues AAWDDSLSGWV (residues 91to 101, comprising CDR-L3), and AREYYDSSGYYHLDY (residues 96 to 110,comprising CDR-H3 residues 98-110 plus two additional amino acidsflanking the CDR at its N-terminal site).

The resulting single NNK libraries were sequenced and 139 amino acidexchange variants of TPP-12387 for CDR-L3 and 156 CDR-H3 were identified(see Table 3.1 and 3.2).

TABLE 3.1 List of exchanges in the CDRL3  of TPP-12387 A91 A92 W93 D94D95 S96 L97 S98 G99 W100 V101 R R A A A A R A R R A D N R N R R D R N NQ D C C N D C N D C E C E Q C C G D C E G G G E E E H C E G H H L G G IK E I I I I K L I M M G L L L L M K L T F I K F M K S S M W P L F S F MT T P Y S M P Y S S Y W S V T W S V W T V V T W Y W D Y Y W V V Y C V VV V E G I L K F

TABLE 3.2 List of exchanges in the CDRH3 plus twoN-terminally adjacent amino acids of  TPP-12387 A96 R97 E98 Y99 Y100D101 S102 S103 R N R A R A R A N Q N R D R N R D G C D G H C N H I L E LI E E L L K G S L G G K S F H W F L L M Y P L V S K K S V T K F M T V MT P V P W T S Y W T V W V G104 Y105 Y106 H107 L108 D109 Y110 A A A C C EC N D R D D F E C G N E E G F E I D K F K G H L Q L G L I L I I M I M LK S L N M S Q M T M Q P T R P W S R S V S S V T S T W V W T W Y X V V W

All variants were expressed by transient transfection of the mammaliancell line HEK293 and resulting expression supernatants were directlytested for their binding ability as well as their function blockingactivity towards human alpha2-Antiplasmin.

Those variants showing higher binding and function blocking activitythan the antibody TPP-12387 were expressed one more time in HEK293cells, the antibodies were purified and quantified as described inExample 2 and re-tested in direct comparison with the parental variantTPP-12387 for their binding ability and their function blocking abilitytowards human alpha2-Antiplasmin.

By this, 5 exchanges within the CDR-L3 and 7 exchanges within CDR-H3were identified with improved activity compared to the parental antibodyTPP-12387. These 11 single substitution variants with improved affinityand functional efficiency were recombined in one recombination librarybased on TPP-12387. Here oligonucleotides were generated to introduceselected mutations or the corresponding wild type amino acid at eachselected position. Library construction was performed using sequentialrounds of overlap extension PCR. The final PCR product was ligated intoa mammalian IgG1 expression vector and variants were sequenced.

Antibodies generated in this recombination library were TPP-14290,TPP-14291, TPP-14292, TPP-14293, TPP-14294, TPP-14295, TPP-14296,TPP-14297, TPP-14298, TPP-14299, TPP-14300, TPP-14301, TPP-14302,TPP-14303, TPP-14304, TPP-14305, TPP-14306, TPP-14307, TPP-14308,TPP-14309, TPP-14310, TPP-14311, TPP-14312, TPP-14313, TPP-14314,TPP-14315, TPP-14316, TPP-14317, TPP-14318, TPP-14319, TPP-14320,TPP-14322, TPP-14323, TPP-14324.

As described in Example 2 antibodies were purified from supernatants andtheir concentration determined. Next, antibodies were tested for theirability of binding human alpha2-Antiplasmin and their ability ofblocking human alpha2-Antiplasmin (as described in Example 3 and 4).

Antibodies TPP-14293, TPP-14298, TPP-14303, TPP-14305, TPP-14308,TPP-14313, TPP-14314, TPP-14318, TPP-14323 represent the most improvedrecombined mutants of TPP-12387 as identified as being most potent interms of binding human alpha2-Antiplasmin and blocking the activity ofhuman alpha2-Antiplasmin. Binding as well as activity data for theseantibodies are given in FIG. 7 .

According to the methods described in Example 3 and Example 4,antibodies TPP-14323, TPP-14318, TPP-14314, TPP-14313, TPP-14308,TPP-14305, TPP-14303, TPP-14298, and TPP-14293 were tested for theirability to bind to and to block the activity of human alpha2-Antiplasminin a dose-dependent manner. For each antibody, one dose response curvefrom two to three independent experiments performed in quadruplicate isshown as example in FIG. 7.1 to FIG. 7.18 . (see Table 3.3)

TABLE 3.3 Binding activity EC [M] and function blocking activity IC50[M] of the three independent experiments binding activity [EC50 M]function blocking activity [IC50 M] antibody 1st exp*. 2nd exp. 3rd exp.1st exp*. 2nd exp. 3rd exp. TPP-14293 1.2E−08 1.2E−08 1.2E−08 4.6E−084.8E−08 5.0E−08 TPP-14298 5.7E−09 6.2E−08 5.8E−08 3.1E−08 3.7E−084.0E−08 TPP-14303 2.4E−08 2.9E−08 2.7E−05 8.5E−08 8.9E−08 9.4E−08TPP-14305 1.5E−08 1.6E−08 1.6E−08 6.4E−08 6.9E−08 6.6E−08 TPP-143085.7E−09 6.1E−08 6.0E−08 2.4E−09 3.0E−08 2.4E−08 TPP-14313 3.3E−093.5E−08 3.4E−08 1.2E−08 2.0E−08 1.3E−08 TPP-14314 8.1E−09 8.2E−088.4E−08 6.8E−08 7.3E−08 6.9E−08 TPP-14318 5.7E−09 5.9E−08 6.1E−082.0E−08 2.5E−08 2.2E−08 TPP-14323 7.0E−09 7.3E−08 7.1E−08 2.0E−082.0E−08 2.9E−08 *Shown in Figure 7

Example 6: Risk Reduction for Sequence-Based Immunogenicity

In order to reduce the risk for sequence-based immunogenicity antibodyTPP-14308 was chosen for further optimization. For this, amino acidswhich differ from the nearest germline sequence were exchanged, thecorresponding cDNAs were synthesized, HEK293 cells were transientlytransfected, the expressed antibodies of this invention were quantifiedbut not purified and tested for their ability to bind humanalpha2-Antiplasmin and to block the function of humanalpha2-Antiplasmin.

Outcome of this approach were 47 antibodies.

Most germline exchanges show only minor improvement in bothfunctionalities.

Although only amino acids within the framework sequences and CDRH1 andCDRH2 have been exchanged, surprisingly, compared to antibody TPP-14308,6 variants were identified exhibiting even higher binding activityand/or improved function blocking activity.

Dose response curves of binding and function blocking activities ofthese variants are given in FIG. 8 .

TABLE 3.5 Binding activity EC [M] and function blocking activity IC50[M] of two to three independent experiments binding activity [EC50 M]function blocking activity [IC50 M] 1 st exp.* 2nd exp. 3rd exp. 1stexp*. 2nd exp. 3rd exp. TPP-17041 1.9E−10 2.3E−10 5.6E−10 6.9E−10TPP-17044 3.7E−11 4.9E−11 1.0E−10 1.7E−10 2.3E−10 2.0E−10 TPP-170458.5E−11 1.2E−10 1.1E−10 4.0E−10 5.0E−10 TPP-17048 1.8E−10 3.6E−102.8E−10 3.9E−10 3.0E−10 TPP-17051 1.1E−10 1.6E−10 2.2E−10 3.3E−104.4E−10 TPP-17053 5.2E−10 5.8E−10 6.3E−10 4.3E−10 6.5E−10 5.0E−10 *Shownin Figure 8

In a next step, the most active antibody TPP-17044 was expressed inlarger amounts and was purified and quantified. The purified andquantified antibody was retested for its function blocking activity ofhuman, cynomolgus, and rabbit alpha2-Antiplasmin. Results are shown inFIG. 9 . One dose response curve from two to three independentexperiments performed in quadruplicate is shown as an example. Functionblocking activity of TPP-17044 for human alpha2-Antiplasmin was 4.4E-10M (as shown in FIGS. 9.1 ), and 5.4E-10 M for the second and 5E-10 M forthe third experiment. For the inhibition of Cynomolgusalpha2-Antiplasmin values were 4.6E-10 M (FIG. 9.2 ), 4.9E-10 M for thesecond experiment and 5.1E-10 M for the third experiment. Rabbitalpha2-Antiplasmin was blocked in its activity by TPP-17044 with IC50values of 2.7E-08 M (FIG. 9.3 ), 3.6E-08 M for a second experiment and2.9E-08 M for a third experiment.

Finally, in order to further minimize the theoretic risk of animmunogenic reaction TPP-17044 was re-cloned into the human IgG4 Fcversion of human antibodies. The resulting antibody TPP-17928 was againtested for its function blocking activity on human, cynomolgus, andrabbit alpha2-Antiplasmin. Results are shown in FIG. 10 . One doseresponse curve from two to three independent experiments performed inquadruplicate is shown as an example. Function blocking activity ofTPP-17928 for human alpha2-Antiplasmin was 1.1E-10 M (as shown in FIGS.10.1 ), and 1.6E-10 M for the second experiment. For the inhibition ofCynomolgus alpha2-Antiplasmin values were 2.6E-10 M (FIG. 10.2 ),3.4E-10 M for the second experiment and 2.9E-10 M for the thirdexperiment. Rabbit alpha2-Antiplasmin was blocked in its activity byTPP-17928 with IC50 values of 1.5E-08 M (FIG. 10.3 ), 1.9E-10 M for asecond experiment and 1.6E-10 M for a third experiment.

Example 7: In Vitro Clot Lysis

Human blood was collected by venipuncture from healthy subjects who hadnot been medicated during the last 10 days (procedure approved by EthicsCommittee “Ärztekammer Nordrhein”, #2017029). Blood was collected intoplastic tubes containing 1/10 volume of 3.8% trisodium citrate.Platelet-Poor Plasma (PPP) was obtained by immediate centrifugation at2500 g for 10 min at 4° C., and stored at −20° C.

For all experiments plasma from at least n=3 independent donors wasused. Clot lysis assay was performed as followed. Frozen plasma wasthawed (at 37° C. for 30 min) and mixed with defined concentrations ofthe test compounds [varying between 0.015 μM-1 μM] or solvent only ascontrol in a 96-well plate. On a second 96-well plate CaCl2 (Sigma;catalogue number 21115-100ML) [final concentration: 12.5 mM] as clotinducer and low dose tPA (Actilyse®, Boehringer Ingelheim) [finalconcentration 0.3 μg/ml] as lysis initiator were prepared. After shortincubation of test compound in plasma (5 min at 37° C.) the mixture wasadded to the second plate and directly transferred to a microplatereader (Tecan infinite 200 Pro) to measure absorption (at 405 nm, 37°C., 1/min) over time for 3 h. For final determination of lysis timereduction tPA-induced lysis time was set as 100% (individually for everydonor plasma) and lysis time reduction was calculated for each testcompound in a dose-response curve.

For analysis of rabbit plasma, whole blood from male New Zealand Whiterabbits was obtained via venipuncture and prepared and used as describedabove for human plasma.

As shown in FIG. 11 , antibody TPP-17928 reduces the tPA-induced clotlysis time in a dose-dependent manner in human and in rabbit plasma,respectively. For human plasma, value for the function blocking activityof TPP-17928 was 2.5E-07 M and for rabbit plasma 2.3E-07 M.

Example 8: In Vivo Testing of TPP-17928 in an Acute Pulmonary Embolismin Rabbits

To study the effects of the anti-anti-alpha2-Antiplasmin antibody ofthis invention on clot dissolution in pulmonary embolism a rabbit invivo model was used.

Male New Zealand White Rabbits were anesthetized by an intramuscularinjection of Xylazine/Ketamine (5 mg/kg+40 mg/kg, Sigma, cataloguenumbers X₁₁₂₆ and K2753). Ears, neck and the left hind limb (in the areaof the femoral triangle) were shaved. To keep the rabbit anesthetized aninfusion of Xylazine/Ketamine (80 ml+800 ml ad 60 ml NaCl 0.9%) with 5ml/h was given via the ear vein. The rabbits were placed on a heatingplate and kept at 37° C. for the whole experimental time. The left venafemoralis was cannulated for compound application and blood sampling,the right vena jugularis for clot injection.

For preparation of fluorescently labeled clots, rabbit platelet poorplasma was mixed with ALEXA 488 fluorescently-labeled human Fibrinogen(Thermo Fisher Scientific, catalogue number F13191). Clotting wasinitiated by adding 2.5 μl Batroxobin [20 U/ml] (LOXO, catalogue number101-04,) and 2.5 μl CaCl2 [0.1 mM] to 45 μl of plasma mixture; finalclot volume 50 μl containing 75 μg of fluorescently-labeled Fibrinogen.

After clot maturation (30 min at 37° C.) 2 clots/kg bodyweight wereinjected into the jugular vein of the anesthetized rabbit which led toembolization of clots in the lung. 30 min after embolization treatmentwas started by bolus i.v. injection of either saline, antibodyTPP-17928, tPA or a combination of antibody TPP-17928 and tPA. Over atime period of 360 min blood samples were taken from the rabbits' veinand plasma fluorescence was analyzed as indirect parameter of clotdissolution. Simultaneously, directly after treatment onset and 300 minpost treatment the ear bleeding time was determined. To access the earbleeding time an incision of app. 3-5 mm was made with a scalpel bladein parallel to the outer edge of the ear (close to the outer ear vein).Every 30 sec it was proven whether the incision was still bleeding bygently dabbing with a small filter tip directly besides the incision.

As shown in FIG. 12.1 , treatment of pulmonary embolism in rabbits withantibody TPP-17928 led to an increased clot dissolution which isconsidered over time comparable to tPA treatment. TPP-17928dose-dependently increases the plasma fluorescence (2.1-fold increase ofAUC in comparison to control) as indirect measurement parameter for clotdissolution. Combination of the 15 mg/kg of TPP-17928 with a lowconcentration of tPA (0,125 mg/kg) accelerates clot dissolution evenfaster. tPA-treatment also shows a dose-dependent effect on clotdissolution (FIG. 12.2 ).

Similar results with an increase of plasma fluorescence as parameter forclot dissolution were observed for 30 mg/kg TPP-12387 (2.6-fold increaseof AUC in comparison to control) and 15 mg/kg TPP-17044 (2.1-foldincrease of AUC in comparison to control) in further rabbit pulmonaryembolism experiments.

Determination of Ear Bleeding Time

In the above described experiment, simultaneously, the ear bleeding timewas determined. Directly after treatment application (at 0 min) the earbleeding time was measured as follows. To access the ear bleeding timean incision of app. 3-5 mm was made with a scalpel blade in parallel tothe outer edge of the ear (close to the outer ear vein). Every 30 sec itwas proven whether the incision was still bleeding by gently dabbingwith a small filter tip directly besides the incision. Ear bleeding timeunravels a superior safety profile for anti-a2AP-antibody treatment incomparison to tPA treatment. Directly after compound administrationthere is no increase in bleeding time detectable for none of the usedantibody concentrations whereas tPA treatment especially at the highestconcentration of 1 mg/kg shows an immediate effect on ear bleeding timeprolongation. Surprisingly, also the combination of the mg/kg ofTPP-17928 in combination with the low dose of tPA (0.125 mg/kg) does notlead to a significant increase in bleeding time. This clearly proofs forsuperiority of the anti-a2AP-antibody treatment over tPA treatment inrespect of adverse events, in this case bleeding. Results are shown inFIG. 13 . Bleeding time prolongation for tPA [1 mg/kg] 2.03-fold, forTPP-17928 [15 mg/kg] 0.95-fold and for the combination of tPA [0.125mg/kg]+TPP-17928 [15 mg/kg] 1.56-fold. Similar results with no effect onear bleeding time prolongation were observed for 30 mg/kg TPP-12387(0.91-fold) and 15 mg/kg TPP-17044 (1.05-fold) in further rabbit earbleeding experiments.

Example 9: Determination of the Binding Affinity of Antibodies of theInvention

Binding assays were performed on a Biacore T200 instrument at 25° C.with a protein G sensor chip and assay buffer HBS-EP+. Antibodies werecaptured to ˜150 RU and analytes were used at concentrations between1.56 and 200 nM for full kinetics. As interaction partner,alpha2-Antiplasmin from human, cynomolgus and rabbit was used.Regeneration was performed with glycine-HCl pH 1.5. Kinetic parameterswere derived by fitting experimental sensorgrams to a 1:1 Langmuirbinding model. Results are given in Table 4.

TABLE 4 Affinity values of antibodies of the invention. Ligand AnalyteK_(D) [M] TPP-12387 Human 1.8 E−07 Rabbit 4.3 E−08 Cyno 1.0 E−07TPP-14308 Human 3.9 E−09 Rabbit 7.8 E−09 Cyno 3.1 E−09 TPP-17041 Human1.1 E−08 Rabbit 1.8 E−08 Cyno 7.6 E−09 TPP-17044 Human 5.1 E−09 Rabbit5.4 E−09 Cyno 3.8 E−09 TPP-17045 Human 1.4 E−08 Rabbit 4.9 E−08 Cyno 1.1E−08 TPP-17048 Human 7.9 E−09 Rabbit 9.7 E−09 Cyno 5.8 E−09 TPP-17051Human 1.8 E−08 Rabbit 1.7 E−08 Cyno 1.3 E−08 TPP-17053 Human 1.1 E−08Rabbit 2.8 E−08 Cyno 8.7 E−09 TPP-17928 Human   1 E−09 Rabbit   5 E−09Cyno   2 E−09

Example 10: Direct Comparison of the Function Blocking Activity of 77A3and of Antibodies of the Invention

In order to compare the blocking activity of the test antibody 77A3 withthe test antibodies of the invention against the A2AP activity thefunction blocking assay as described in detail in example 4 was used. Inbrief, the test antibodies were pre-incubated at a concentration of6.1E-11-3.0E-08 M and 0.0E-06 M, respectively with A2AP. After addingplasmin and 1-1275 (a fluorogenic substrate for plasmin serine proteaseactivity) to the assay the fluorescence signal as a measure for theserine protease activity of plasmin was determined.

An increase of 77A3 concentration up to 0.03 μM resulted into anincrease of fluorescence signal due to the cleavage of the fluorogenicsubstrate 1-1275 by plasmin. However, a further increase in 77A3concentration resulted in a decrease of fluorescence signal. Thatindicates that, testing the antibody 77A3 in the biochemical assaydescribed in Example 4 up to a concentration of 0.03 μM leads toblockade of alpha2-Antiplasmin and that a further increase in 77A3antibody concentration leads to a decline in plasmin activity, resultingin a complete inhibition of plasmin activity at a 77A3 concentration of1 μM (FIG. 14.1 ). For the antibodies of the invention, the followingIC50 values for the function blocking activity of A2AP were generated:TPP-17041-6.4 E-10 M, TPP-17044-3.9 E-10 M, TPP-17045-1.4 E-10 M,TPP-17048-1.2 E-09 M, TPP-17051-3.3 E-10 M, TPP-17053-1.5 E-10 M. Noneof the antibodies of the invention tested up to a concentration of 1 μMresults in a decrease of fluorescence signal indicating that testingantibodies of the invention have no impact on plasmin activity (FIG.14.2-14.7 ).

Example 11: Biochemical Assay for Testing Antibodies of the Inventionfor Inhibiting the Proteolytic Activity of Plasmin

To further investigate the surprising results obtained for the antibody77A3 in Example 10 anti-alpha2-Antiplasmin antibodies of the inventionas well as the antibody 77A3 were tested for inhibiting activity in abiochemical Plasmin assay. For this, different concentrations ofantibodies starting at a defined concentration, followed by 1:2 or 1:3dilution steps, were incubated for 1 hour at 37° C. with 400 pM of humanPlasmin obtained from Haematologic Technologies INC.; catalogue numberHCPM0140) and the fluorogenic substrate 1-1275 (Bachem;MeOSuc-Ala-Phe-Lys-AMC trifluoroacetate salt; catalogue number 1-1275(stock: 10 mM in DMSO)) at a final concentration of 1 μM. This reactionwas carried out in 384 well microtiter plates (Nunc; catalogue number262260). The fluorogenic signals were measured at the followingconditions: modus fluorescence top reading, Ex 360 nM, Em 465 nm, Exbandwidth 5 nm, Em bandwidth 5 nm.

As shown in FIG. 16 , TPP-17928 (exemplarily shown for all antibodies ofthe invention) has no impact on the biochemical activity whereas 77A3blocks the proteolytic activity of Plasmin with an IC50 value of 1.7 μM.

TABLE 5 Inhibition of plasmin proteolytic activity by 77A3 and testantibodies of the invention. antibody IC₅₀ [M] 77A3*   1.7 E−06TPP-12387 >1.0 E−05 TPP-14293 >5.0 E−06 TPP-14298 >5.0 E−06TPP-14303 >1.0 E−05 TPP-14305 >2.0 E−06 TPP-14308 >5.0 E−06TPP-14313 >5.0 E−06 TPP-14314 >2.0 E−06 TPP-14318 >1.0 E−05TPP-14323 >1.0 E−5 TPP-17041   >1 E−05 TPP-17044 >1.0 E−05TPP-17045 >1.0 E−05 TPP-17048 >1.0 E−05 TPP-17051 >1.0 E−05TPP-17053 >1.0 E−05 TPP-17928* >1.0 E−05 *dose response curve from twoto three independent experiments performed in duplicates are shown asexample in FIG. 16 for 77A3 and TPP-17928.

Example 12: Epitope Mapping of TPP-12387 and 77A3

Epitope mapping was performed by the company PEPperPRINT (Heidelberg,Germany) by using the PEPperCHIP® Peptide Microarray platform.

For linear epitope mappings, the antigen sequence was translated intooverlapping linear 15 amino acid peptides with a peptide-peptide overlapof 14 amino acids. The resulting peptide microarrays contained 491different peptides printed in duplicate.

For the conformational epitope mappings, the antigen sequence wastranslated into overlapping 7, 10 and 13 amino acid peptides withpeptide-peptide overlaps of 6, 9 and 12 amino acids. After peptidesynthesis, all peptides were cyclized via a thioether linkage between aC-terminal cysteine side chain and an appropriately modified N-terminus.The resulting conformational peptide microarrays contained 1,488different cyclic constrained peptides printed in duplicate.

Microarray were blocked by using Rockland blocking buffer MB-070 (30 minbefore the first assay), antibody incubation was performed in Incubationbuffer consisting of PBS, pH 7.4 with 0.05% (linear epitope mappings) or0.005% (conformational epitope mappings) Tween 20 with 10% blockingbuffer.

Following incubation, arrays were washed using PBS, pH 7.4 with 0.05%(linear epitope mappings) or (conformational epitope mappings) Tween 20.Arrays were washed 3×1 min (linear epitope mappings) or 2×10 sec(conformational epitope mappings).

Antibodies were incubated on the Microarray in concentrations of 1μg/ml, 10 μg/ml and 100 μg/ml in incubation buffer. Incubation time was16 h at 4° C. and shaking at 140 rpm.

As control, the mouse monoclonal anti-HA (12CA5) DyLight800 was used ata dilution of 1:2000; this detection antibody was incubated on theMicroarray at 45 min staining in incubation buffer at room temperature.

As secondary antibody the goat anti-human IgG (Fc) DyLight680 was usedat a dilution of 1:5000. This detection antibody was incubated on theMicroarray at 45 min staining in incubation buffer at room temperature.

Signals were detected by using the LI-COR Odyssey Imaging System with ascanning offset 0.65 mm, a resolution 21 μm, and scanning intensities of7/7 (red=700 nm/green=800 nm).

Quantification of spot intensities and peptide annotation were based onthe 16-bit gray scale tiff files at scanning intensities of 7/7 thatexhibit a higher dynamic range than the 24-bit colorized tiff files.Microarray image analysis was done with PepSlide® Analyzer. A softwarealgorithm was used to break down fluorescence intensities of each spotinto raw, foreground and background signal, and calculated averagedmedian foreground intensities and spot-to-spot deviations of spotduplicates. Based on averaged median foreground intensities, anintensity map was generated and interactions in the peptide maphighlighted by an intensity color code with red for high and white forlow spot intensities. Averaged spot intensities of the assays wereplotted with the antibody samples against the antigen sequence from theN- to the C-terminus to visualize overall spot intensities andsignal-to-noise ratios. The intensity plots were correlated with peptideand intensity maps as well as with visual inspection of the microarrayscans to identify the epitopes of the tested antibodies.

Pre-staining of a linear and a conformational peptide microarray withsecondary goat anti-human IgG (Fc) DyLight680 antibody (1:5000) andcontrol mouse monoclonal anti-HA (12CA5) DyLight800 antibody (1:2000)did not show any background interaction with the linear or cyclicconstrained peptides of the antigen. In contrast incubation with theantibodies of interest resulted in the following observations:

As shown in table 6, TPP-12387 showed a high signal-to-noise ratioagainst peptides with the consensus motif SRMSLSS, which corresponds toamino acid 402-408 of SEQ ID NO: 1 which is located in the reactivecenter loop of A2AP (amino acid 400-412 of Seq ID NO: 1).

5 In contrast, for the antibody 77A3, a very weak signal-to-noise ratioagainst peptides with the basic consensus motif RPTKVRLPK wasidentified, that corresponds to amino acid 330-338 of SEQ ID NO: 1. Forthis part of A2AP no distinct have been described.

TABLE 6 A2AP binding sites for TPP-12387 and 77A3 signal- signal-signal- to-noise- to-noise- to-noise-  peptide  ratio at   ratio atratio at sequence 1 μg/ml 10 μg/ml 100 μg/ml TPP- SRMSLSS 659.0 2,255.012387 77A3 RPTKVRLPK  11.5    40.0 134.0

What is claimed is:
 1. An isolated antibody or antigen-binding fragmentthereof capable of binding to A2AP and inhibiting the activity of A2AP,wherein said isolated antibody or antigen-binding fragment thereof doesnot inhibit plasmin activity.
 2. The isolated antibody orantigen-binding fragment thereof according to claim 1, wherein saidisolated antibody or antigen-binding fragment thereof binds to humanA2AP of the sequence of amino acid 40-491 of SEQ ID NO: 1 with adissociation constant (K_(D)) selected from the group consisting of ≤100nM, ≤50 nM, ≤25 nM, ≤10 nM, ≤1 nM, and ≤0.5 nM; and wherein saidisolated antibody or antigen-binding fragment thereof inhibits theactivity of human A2AP of the sequence of amino acid 40-491 of SEQ IDNO: 1 with an EC₅₀ selected from the group consisting of ≤500 nM, ≤250nM, ≤100 nM, ≤50 nM, ≤25 nM, ≤10 nM, ≤1 nM, and ≤0.5 nM in an in vitroA2AP function blocking assay.
 3. The isolated antibody orantigen-binding fragment thereof according to claim 1, wherein saidisolated antibody or antigen-binding fragment thereof does not inhibitplasmin activity up to a concentration of said isolated antibody orantigen-binding fragment thereof selected from the group consisting of 1μM, 2 μM, 5 μM, and 10 μM in an in vitro plasmin inhibition assay. 4.The isolated antibody or antigen-binding fragment thereof according toclaim 1, wherein said plasmin is human plasmin comprising SEQ ID NO: 118and SEQ ID NO:
 119. 5. The isolated antibody or antigen-binding fragmentthereof according to claim 1, comprising: i) a heavy chainantigen-binding region that comprises a heavy chain complementaritydetermining region 1 (H-CDR11 comprising SEQ ID NO: 6, an H-CDR2comprising SEQ ID NO: 8, and an H-CDR3 comprising SEQ ID NO: 13 and alight chain antigen-binding region that comprises a light chaincomplementarity determining region 1 (L-CDR11 comprising SEQ ID NO: 9,an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ IDNO:18; ii) a heavy chain antigen-binding region that comprises an H-CDR1comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and anH-CDR3 comprising SEQ ID NO: 13 and a light chain antigen-binding regionthat comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprisingSEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18; iii) a heavy chainantigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6,an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO:11 and a light chain antigen-binding region that comprises an L-CDR1comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and anL-CDR3 comprising SEQ ID NO:17; iv) a heavy chain antigen-binding regionthat comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprisingSEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 11 and a light chainantigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9,an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ IDNO:18; v) a heavy chain antigen-binding region that comprises an H-CDR1comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and anH-CDR3 comprising SEQ ID NO: 12 and a light chain antigen-binding regionthat comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprisingSEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18; vi) a heavy chainantigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6,an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO:13 and a light chain antigen-binding region that comprises an L-CDR1comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and anL-CDR3 comprising SEQ ID NO:19; vii) a heavy chain antigen-bindingregion that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 14 and alight chain antigen-binding region that comprises an L-CDR1 comprisingSEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3comprising SEQ ID NO:18; viii) a heavy chain antigen-binding region thatcomprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ IDNO: 7, and an H-CDR3 comprising SEQ ID NO: 14 and a light chainantigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9,an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ IDNO:20; ix) a heavy chain antigen-binding region that comprises an H-CDR1comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and anH-CDR3 comprising SEQ ID NO: 15 and a light chain antigen-binding regionthat comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprisingSEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18; x) a heavy chainantigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6,an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO:16 and a light chain antigen-binding region that comprises an L-CDR1comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and anL-CDR3 comprising SEQ ID NO:18; xi) a heavy chain antigen-binding regionthat comprises an H-CDR1 comprising SEQ ID NO: 21, an H-CDR2 comprisingSEQ ID NO: 8, and an H-CDR3 comprising SEQ ID NO: 13 and a light chainantigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9,an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ IDNO:18; or xii) a heavy chain antigen-binding region that comprises anH-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 22, andan H-CDR3 comprising SEQ ID NO: 13 and a light chain antigen-bindingregion that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18.
 6. Theisolated antibody or antigen-binding fragment thereof according to claim1, comprising: xiii) a variable heavy chain domain comprising SEQ ID NO:32 and a variable light chain domain comprising SEQ ID NO: 38; xiv) avariable heavy chain domain comprising SEQ ID NO: 23 and a variablelight chain domain comprising SEQ ID NO: 37; xv) a variable heavy chaindomain comprising SEQ ID NO: 24 and a variable light chain domaincomprising SEQ ID NO: 38; xvi) a variable heavy chain domain comprisingSEQ ID NO: 25 and a variable light chain domain comprising SEQ ID NO:38; xvii) a variable heavy chain domain comprising SEQ ID NO: 26 and avariable light chain domain comprising SEQ ID NO: 38; xviii) a variableheavy chain domain comprising SEQ ID NO: 27 and a variable light chaindomain comprising SEQ ID NO: 39; xix) a variable heavy chain domaincomprising SEQ ID NO: 27 and a variable light chain domain comprisingSEQ ID NO: 38; xx) a variable heavy chain domain comprising SEQ ID NO:28 and a variable light chain domain comprising SEQ ID NO: 38; xxi) avariable heavy chain domain comprising SEQ ID NO: 28 and a variablelight chain domain comprising SEQ ID NO: 40; xxii) a variable heavychain domain comprising SEQ ID NO: 29 and a variable light chain domaincomprising SEQ ID NO: 38; xxiii) a variable heavy chain domaincomprising SEQ ID NO: 30 and a variable light chain domain comprisingSEQ ID NO: 38; xxiv) a variable heavy chain domain comprising SEQ ID NO:31 and a variable light chain domain comprising SEQ ID NO: 38; xxv) avariable heavy chain domain comprising SEQ ID NO: 33 and a variablelight chain domain comprising SEQ ID NO: 38; xxvi) a variable heavychain domain comprising SEQ ID NO: 34 and a variable light chain domaincomprising SEQ ID NO: 38; xxvii) a variable heavy chain domaincomprising SEQ ID NO: 35 and a variable light chain domain comprisingSEQ ID NO: 38; or xxviii) a variable heavy chain domain comprising SEQID NO: 36 and a variable light chain domain comprising SEQ ID NO:
 38. 7.The isolated antibody or antigen-binding fragment thereof according toclaim 1, wherein the isolated antibody is an IgG antibody.
 8. Theisolated antibody according to claim 7 comprising: xxix) a heavy chaincomprising SEQ ID NO: 55 and a light chain comprising SEQ ID NO: 57;xxx) a heavy chain comprising SEQ ID NO: 41 and a light chain comprisingSEQ ID NO: 56; xxxi) a heavy chain comprising SEQ ID NO: 42 and a lightchain comprising SEQ ID NO: 57; xxxii) a heavy chain comprising SEQ IDNO: 43 and a light chain comprising SEQ ID NO: 57; xxxiii) a heavy chaincomprising SEQ ID NO: 44 and a light chain comprising SEQ ID NO: 57;xxxiv) a heavy chain comprising SEQ ID NO: 45 and a light chaincomprising SEQ ID NO: 58; xxxv) a heavy chain comprising SEQ ID NO: 45and a light chain comprising SEQ ID NO: 57; xxxvi) a heavy chaincomprising SEQ ID NO: 46 and a light chain comprising SEQ ID NO: 57;xxxvii) a heavy chain comprising SEQ ID NO: 46 and a light chaincomprising SEQ ID NO: 59; xxxviii) a heavy chain comprising SEQ ID NO:47 and a light chain comprising SEQ ID NO: 57; xxxix) a heavy chaincomprising SEQ ID NO: 48 and a light chain comprising SEQ ID NO: 57; xl)a heavy chain comprising SEQ ID NO: 49 and a light chain comprising SEQID NO: 57; xli) a heavy chain comprising SEQ ID NO: 50 and a light chaincomprising SEQ ID NO: 57; xlii) a heavy chain comprising SEQ ID NO: 51and a light chain comprising SEQ ID NO: 57; xliii) a heavy chaincomprising SEQ ID NO: 52 and a light chain comprising SEQ ID NO: 57;xliv) a heavy chain comprising SEQ ID NO: 53 and a light chaincomprising SEQ ID NO: 57; or xlv) a heavy chain comprising SEQ ID NO: 54and a light chain comprising SEQ ID NO:
 57. 9. The isolated antibody orantigen-binding fragment thereof according to claim 1, wherein theantigen-binding fragment is selected from the group consisting of scFv,Fab, Fab′ fragment, and a F(ab′)2 fragment.
 10. The isolated antibody orantigen-binding fragment thereof according to claim 1, which is amonoclonal antibody or antigen-binding fragment thereof.
 11. Theisolated antibody or antigen-binding fragment according to claim 10,which is a human, humanized or chimeric antibody or antigen-bindingfragment thereof.
 12. An isolated antibody or antigen-binding fragmentthereof that competes with the isolated antibody or antigen-bindingfragment thereof according to claim 1 for binding to A2AP.
 13. Anantibody conjugate, comprising the isolated antibody or antigen bindingfragment thereof according to claim
 1. 14. An isolated nucleic acidsequence that encodes the isolated antibody or antigen-binding fragmentthereof according to claim
 1. 15. A vector comprising the nucleic acidsequence according to claim
 14. 16. An isolated cell expressing theisolated antibody or antigen-binding fragment thereof according to claim1, a nucleic acid encoding the isolated antibody or antigen-bindingfragment thereof, a vector comprising the nucleic acid, or a combinationthereof.
 17. (canceled)
 18. A method of producing the isolated antibodyor antigen-binding fragment according to claim 1, comprising culturingof a cell expressing the isolated antibody or antigen-binding fragmentthereof, a nucleic acid encoding the isolated antibody orantigen-binding fragment thereof, a vector comprising the nucleic acid,or a combination thereof and purifying the antibody or antigen-bindingfragment thereof.
 19. A pharmaceutical composition comprising theisolated antibody or antigen-binding fragment thereof according claim 1or an antibody conjugate comprising the isolated antibody or antigenbinding fragment thereof, and one or more pharmaceutically acceptableexcipients.
 20. (canceled)
 21. (canceled)
 22. A method for the treatmentor prophylaxis of disorders or diseases associated with ischemic eventsdue to partial or complete vessel occlusion, the method comprisingadministration of a therapeutically effective amount of the isolatedantibody or antigen-binding fragment thereof according to claim
 1. 23.(canceled)
 24. A kit comprising the isolated antibody or antigen-bindingfragment thereof according to claim 1, an antibody conjugate comprisingthe isolated antibody or antigen binding fragment thereof, or thepharmaceutical composition thereof and instructions for use.